ISRN Chromatography

Volume 2012 (2012), Article ID 894965, 8 pages

doi:10.5402/2012/894965

Research Article

Development and Validation of RP-HPLC Method for the Determination of Ganciclovir in Bulk Drug and in Formulations

P. J. Ramesh, K. Basavaiah, K. B. Vinay, and Cijo M. Xavier

Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India

Received 1 April 2012; Accepted 9 May 2012

Academic Editors: E. Boselli, F. M. Lancas, M. P. Marszall, and S. Sarker

Copyright © 2012 P. J. Ramesh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A simple, rapid, accurate, and precise gradient reversed-phase HPLC (RP-HPLC) method has been developed for the determination of ganciclovir (GNC) in pharmaceuticals. Chromatographic separation was carried out on inertsil ODS C₁₈ (4.6 mm  mm, 5.0 μm) LC column using ammonium acetate buffer, sodium salt of hexane sulfonic acid as ion-pairing reagent in 1000 mL water, and acetonitrile (90 : 10) (v/v) as mobile phase at a flow rate of 1.0 mL  and with UV detection at 245 nm at column temperature (30°C). The runtime under these chromatographic conditions was 10 min. The method was linear over the range of 0.02–75 μg . The limits of detection (LOD) and quantification (LOQ) values were 4.1 and 20 ng , respectively. The method was successfully extended to study the effect on GNC upon treatment with 2 N NaOH, 2N HCl, and 5% H₂O₂ for 2 hrs at 80°C and upon exposure to UV (1200 K lux hrs) for 72 hrs and thermal (105°C) for 5 hrs. The proposed method was further applied to the determination of GNC in pharmaceuticals, with good percent recovery. The accuracy and the precision of the method were validated on intraday and interday basis in accordance with ICH guidelines.

1. Introduction

Ganciclovir (GNC), chemically known as 2-amino-9--6,9-dihydro-3H-purin-6-one (Figure 1), is a nucleoside analogue widely used in the treatment of cytomegalovirus infections.

Figure 1: Structure of ganciclovir.

It has proved to be effective against cytomegalovirus in immunocompromised patients, mainly in those with the acquired immunodeficiency syndrome (AIDS), congenital immunodeficiency, or in individuals following organ transplantation [1, 2]. Various techniques have been developed for the determination of GNC in pharmaceuticals. It is official in the United States Pharmacopoeia [3], which describes an HPLC method for its determination in injections and in oral suspension.

The literature is enriched with several methods for the determination of GNC in pharmaceutical dosage forms including body fluids. The most extensively used technique for the quantitation of ganciclovir is HPLC, but most of the procedures using this technique are devoted to body fluids like plasma [4–14], plasma and tissues [15], serum [16], and blood samples [17]. There is only one report [18] dealing with the application of HPLC for the determination of pharmaceutical formulations, that is eye drops. GNC in bulk drug and in its formulations has been assayed by UV spectrometry by measuring the absorbance of 0.1 M HCL and 0.1 M NaOH at 253 and 266 nm, respectively [19]. The methods reported are moderately sensitive with molar absorptivity values of . Other reported methods for pharmaceuticals include visible spectrophotometry [20–22], flow injection luminescence spectrometry [23], and radioimmunology [24–26]. The aim of this study was to develop an analytical HPLC method for sensitive, specific, and rapid determination of GNC in its tablets.

2. Materials and Methods

2.1. Materials and Standards

HPLC-grade acetonitrile (Labscan Asia Co. Ltd., Bangkok, Thailand), analytical reagent grade ammonium acetate (Rankem, Bangalore, India), and sodium salt of hexane sulfonic acid (Rankem, Bangalore, India) were used. Deionised, Milli Q water (Millipore, Bangalore, India) was used to prepare the mobile phase. Ganciclovir was obtained from Lotus Pharmaceuticals Ltd., Bangalore, India. Tablets were purchased from local commercial sources. A stock-standard solution equivalent to 1000 μg mL⁻¹ GNC was prepared by dissolving accurately weighed amount of pure drug in the mobile phase.

2.2. Equipment

HPLC analysis was performed on an Alliance Waters HPLC system equipped with Alliances 2657 series low-pressure quaternary pump, a programmable variable wavelength UV-visible detector, Waters 2996 photodiode array detector, and auto sampler. Data were collected and processed using Waters Empower 2.0 software.

2.3. Chromatographic Conditions

Chromatographic analysis was carried out on an inertsil ODS-3V C₁₈ (4.6 mm i.d 250 mm, 5.0 μm) LC column. The mobile phase consisted of 0.025 M ammonium acetate (pH6.8) containing 0.4 g sodium hexane sulphonate and acetonitrile (90 : 10 v/v). The mobile phase, filtered through 0.45 μm membrane filter and degassed before use, was pumped at 1 mL min⁻¹ flow rate. The column was thermostated at 30°C. Under these conditions the runtime was 10 min.

3. Procedures

3.1. Calibration Graph

Ten μL of working standard solutions (0.02–75 μg mL⁻¹ GNC) was injected automatically onto the column in triplicate and the chromatograms were recorded. The calibration graph was prepared by plotting the mean peak area percentage versus concentration in μg mL⁻¹ (Figure 2).

Figure 2: Linearity graph.

3.2. Pharmaceutical Preparations

Twenty tablets were accurately weighed and crushed into a fine powder and mixed using a mortar and pestle. A quantity of tablet powder equivalent to 100 mg of GNC was weighed accurately into a 100 mL calibrated flask, 50 mL of mobile phase as diluent added and was sonicated for 20 min to complete dissolution of the GNC, and the solution was then diluted to the mark with the diluent and mixed well. A small portion of the tablet solution (say 10 mL) was withdrawn and filtered through a 0.2 μm filter to ensure the absence of particulate matter. The filtrate was appropriately diluted with the diluent before injection into the column.

3.3. Results and Discussion

Mobile phases consisting of several buffering systems such as phosphoric acid, sodium acetate, potassium dihydrogen orthophosphate, sodium dihydrogen ortho phosphate or ammonium acetate with acetonitrile, methanol, and isopropanol as organic modifiers in different volumetric ratios failed to meet the required system parameters. Finally, the mobile phase consisting of 2.0 g ammonium acetate and 0.40 g of sodium salt of hexane sulphonic acid in 1000 mL water and acetonitrile (90 : 10, v/v) was found to give better theoretical plates (>2000) and tailing factor peak (<1.2). The analysis was carried out at 30°C, which besides being economical, offers many advantages like low-column back pressure, good chromatographic peak shape, improved column efficiency (1.14 tailing factor), higher theoretical plates (4732), and consistency in retention time. Under the stated chromatographic conditions, the mean retention time was 3.183 min (). A model chromatogram is shown in Figure 3. Further, the optimized chromatographic conditions were used to study the effect on GNC after treatment with various stress conditions. Upon treatment with 2 N NaOH, 2 N HCl, and 5% H₂O₂, for 2 hrs at 80°C, separately, there was no change in the retention time but there was a slight change in mean peak area in both 2 N HCl (Figure 4) and 2 N NaOH (Figure 5), and there was a significant change in both peak areas but no change in retention time in 5% H₂O₂ (Figure 6). There was no effect upon exposure to UV at 1200 K lux (Figure 7) and thermal treatment at 105°C (Figure 8), for 5 hrs.

Figure 3: Typical chromatogram (pure GNC, 50 μg mL⁻¹).

Figure 4: Chromatogram after forced degradation with 2 N HCl at 80°C for 2 hrs (pure GNC, 50 μg mL⁻¹).

Figure 5: Chromatogram after forced degradation with 2 N NaOH at 80°C for 2 hrs (pure GNC, 50 μg mL⁻¹).

Figure 6: Chromatogram after forced degradation with 5% H₂O₂ at 80°C for 2 hrs (pure GNC, 50 μg mL⁻¹).

Figure 7: Chromatogram after exposure to UV at 1200 K lux hrs for 72 hrs (pure GNC, 50 μg mL⁻¹).

Figure 8: Chromatogram after thermal treatment at 105°C for 5 hrs (Pure GNC, 50 μg mL⁻¹).

4. Method Validation

4.1. Linearity

Working standard solution of GNC (1000 μg mL⁻¹) was appropriately diluted with the diluent solution to obtain solutions in the concentration range 0.02–75 μg mL⁻¹ GNC. Ten μL of each solution was injected in triplicate onto the column under the operating chromatographic conditions described above. Calibration curve was constructed by plotting the mean peak area percentage versus concentration. The least squares method was used to calculate the slope , intercept , and the correlation coefficient of the regression line. The relation between mean peak area and concentration, expressed by the equation , was linear. Values of slope, intercept, and correlation coefficient () were 59682.7532, −295296.2843, and 0.9999, respectively. A plot of log peak area versus log concentration was a straight line with the slope of 0.9969 indicating excellent linearity between mean peak area and concentration in the range 0.02–75 μg mL⁻¹ GNC as shown in Table 1.

Table 1: Regression and sensitivity parameters.

Parameters
Value

Linearity range, μg mL⁻¹
0.02–75

Slope (b)
59682.7532

Intercept (a)
−295296.2843

Standard deviation of intercept ()
235822.5161

Standard deviation of Slope ()
11483.1236

Correlation coefficient (r)
0.9999

Limit of detection (LOD, ng mL⁻¹)
4.1

Limit of quantification (LOQ, ng mL⁻¹)
20

**, where is the area and is the concentration in μg mL⁻¹.

4.2. Limits of Detection (LOD) and Quantification (LOQ)

LOD and LOQ were estimated from the signal-to-noise ratio. The LOD defined as the lowest concentration that gave a peak area with signal-to-noise ratio between 2-3 was found to be 4.1 ng mL⁻¹. The lowest concentration that provided a peak area with a signal-to-noise ratio 9.68, which is called LOQ, was found to be 20 ng mL⁻¹.

4.3. Specificity

Method specificity was checked by comparing the chromatograms obtained for pure GNC solution, synthetic mixture, tablet solution, and placebo blank. An examination of the chromatograms of the above solutions shown in Figures 3, 9, 10, and 11 reveals the absence of peaks due to additives present in tablet preparations.

Figure 9: Chromatogram of synthetic mixture (GNC, 50 μg mL⁻¹).

Figure 10: Chromatogram of tablet solution (GNC, 50 μg mL⁻¹).

Figure 11: Chromatogram of placebo blank.

4.4. Precision and Accuracy

Method precision was determined from the results of six independent determinations of GNC at three different concentrations, 25, 50, and 75 μg mL⁻¹ on the same day. The interday and intraday relative standard deviation (RSD) values for peak area and retention time for the selected concentrations of GNC were less than 2.0 and 0.5%, respectively. The method accuracy, expressed as relative error (%), was determined by calculating the percent deviation found between concentrations of GNC injected and concentrations found from the peak area. This study was performed by taking the same three concentrations of GNC used for precision estimation. The intraday and interday accuracy (expressed as %RE) values which are <2.9% are also compiled in Table 2.

Table 2: Intraday and interday accuracy and precision.

GNC
Intraday accuracy and precision
Interday accuracy and precision

Injected,
GNC founda,
RE,
RSDb,
RSDc,
GNC founda,
RE,
RSDb,
RSDc,

μg mL⁻¹
μg mL⁻¹
%
%
%
μg mL⁻¹
%
%
%

25.0
25.4
1.6
0.85
0.06
25.3
1.2
1.12
0.33

50.0
50.9
1.8
0.20
0.25
51.0
2.0
0.86
0.42

75.0
76.1
1.5
0.62
0.16
77.2
2.9
1.14
0.26

aMean value of seven determinations, bbased on peak area, cbased on retention time.

4.5. Robustness

To determine the robustness of the method, small deliberate changes in the chromatographic conditions like flow rate and column temperature were made, and the results were compared with the original chromatographic conditions. The results indicated that changing the flow rate (±0.1 mL) had some effect on the chromatographic behavior of GNC. However, the alteration in the column temperature (±2°C) had no significant effect. The results of this study expressed as %RSD are summarized in Table 3.

Table 3: Results of robustness study (GNC concentration, 50 μg mL⁻¹, ).

Chromatographic condition
Modification
Peak area precision ()
Retention time precision ()

Area
Mean area ± SD
Standard error of mean
RSD
Retention time
Mean   ± SD
Standard error
RSD

(%)
(min)
(min)
(min)
(%)

3035118

3.205

0.9
3023146
3025561.667 ± 8606.627
4968.898
0.28
3.201
3.206 ± 0.006
0.004
0.19

3018421

3.213

2994748

3.197

Flow (mL)
1.0
2894621
2972593.667 ± 69592.520
40178.119
2.31
3.198
3.193 ± 0.008
0.005
0.25

3028412

3.184

2791126

2.968

1.1
2890061
2815133.333 ± 66269.856
38259.833
2.35
2.967
2.964 ± 0.007
0.004
0.23

2764213

2.956

2782269

3.108

28
2716213
2757572.000 ± 36042.551
20808.585
1.31
3.107
3.106 ± 0.003
0.002
0.10

2774234

3.102

2994748

3.197

Column temperature (°C)
30
2894021
2972396.667 ± 69932.300
40374.286
2.35
3.192
3.193 ± 0.004
0.002
0.11

3028421

3.190

2802618

3.009

32
2803461
2822807.000 ± 34240.909
19768.436
1.21
3.010
3.010 ± 0.002
0.001
0.05

2862342

3.012

4.6. System Suitability

System suitability parameters were measured to verify the system performance and the values of retention time, number of theoretical plates and tailing factor were , 4732 per column and 1.14, respectively. All the values were within the acceptable range.

4.7. Solution Stability

The stability of standard and sample solutions was determined by monitoring the peak area and retention time over a period of 24 hrs by injecting the solutions at the intervals of 0, 8, 16, and 24 hrs. The standard and sample solutions were stored at ambient temperature (25°C) and protected from light during the stability study. No changes in drug concentrations were observed over a period of 24 hrs as shown by the small %RSD values. The %RSD for peak area () was 1.93% for pure drug solution and the value for retention time () was 1.43%. The results are presented in Table 4. No significant changes in concentration of the active ingredient were observed in the tablet solution as well.

Table 4: Solution stability (GNC concentration was 50 μg mL⁻¹).

Time, hrs
Peak area
Mean ± SD
%RSD
Retention time (min)
Mean ± SD
%RSD

2989840

3.195

0
2895622
2929008 ± 52766
1.80

2901562

3.112

2845671

3.158

8
2859782
2831673 ± 37143
1.31

2789565

3.115

2946830

3.118

16
2985633
2936679 ± 54740
1.86

2877574

3.148

2908480

3.149

24
2858942
2855884 ± 54189
1.90

2800231

3.106

4.8. Method Application

The developed and validated method was successfully applied to determine GNC in tablets. The results obtained tallied closely with the labeled amount in the case of tablets (Table 5), thus indicating the utility of the method for content uniformity evaluation. The results were statistically compared with those obtained by reported method [19] for accuracy and precision by applying the Student’s -test and variance ratio -test. The reference method consisted of the measurement of the tablet extracts absorbance at 253 nm. The calculated - and -values were less than the tabulated values of 2.77 and 6.39 at the 95% confidence level and for five degrees of freedom suggesting that there was significant difference between the proposed method and the reference method with respect to accuracy and precision.

Table 5: Results of determination of GNC in tablets and statistical comparison with the reference method.

Tablet brand name**
Nominal amount, mg
Found* (percent of label claim ± SD)

Reference method
Proposed method
Student’s -value (2.77)
-value (6.39)

Ganguard¹
500
98.51 ± 1.05
99.62 ± 1.85
1.05
3.10

Natclovir²
250
99.06 ± 0.88
100.1 ± 1.61
2.31
3.34

*Mean value of five determinations, **marketed by: ¹Ranbaxy Lab, Ahamedabad, India, ²Natco Pharma Ltd., Pune, India, figures in the parenthesis are the tabulated values for four degree of freedom at 95% confidence level.

4.9. Recovery Study

To the preanalyzed tablet powder, pure GNC was added at three levels and the total was found by the proposed method. Each measurement was triplicated. When the test was performed on 500 mg and 250 mg tablets, the percent recovery of pure GNC was 106.15 and 102.10 with standard deviation of 0.86 and 0.46, respectively. The results indicate that the method is very accurate and that common excipients found in tablet preparations do not interfere and the results are summarized in Table 6.

Table 6: Results of recovery study by standard addition method.

Tablet
GNC in tablet, μg mL⁻¹
Pure GNC added, μg mL⁻¹
Total found, μg mL⁻¹
Pure GNC recovered*, percent ± SD

26
12.5
39.12
101.90 ± 0.16

Ganguard 500 mg
26
25
52.05
100.55 ± 0.72

26
37.5
64.18
106.15 ± 0.86

24.9
12.5
38.03
101.30 ± 0.64

Natclovir 250 mg
24.9
25
51.88
102.10 ± 0.46

24.9
37.5
62.88
100.10 ± 0.22

*Mean value of three determinations.

5. Conclusions

Very simple, cost effective, accurate, and precise RP-HPLC method for determining ganciclovir was developed and validated as per the ICH guidelines Q2 (2005). Analysis was carried at ambient temperature. Besides, short retention time (3.117), runtime (10 min), and flow rate (1 mL min⁻¹) make the method attractive since these features help in saving cost and time of analysis. Ammonium acetate has many properties which are particularly interesting for use on a general purpose buffer [27] the most significant being low toxicity and cost, easy availability, good buffering capacity in the chosen pH range, and ability to provide greatly improved separations without column deterioration. In summary, this method is selective, sensitive, rapid, and reproducible for ganciclovir in its tablets. The precision and accuracy are within acceptable ranges, the quantification limit is as low as 20 ng mL⁻¹ [27], and finally the assay is robust and rugged.

Acknowledgments

Authors thank Lotus Pharmaceuticals Ltd., Bangalore, India for providing the pure sample of ganciclovir as gift. Three of the authors (P. J. Ramesh, K. B. Vainay, and C. M. Xavier) thank the authorities of the University of Mysore, Mysore, for permission and facilities. The same authors are indebted to the Jubilant Lifesciences Ltd., Nanjangud, Mysore, India, for their kind permission to pursue Ph.D. degree programme.

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