INTRODUCTION:

Didanosine (Figure 1) is chemically named as 9-[(2R, 5S)-5-(hydroxyl methyl)oxolan-2-yl]-6,9-dihydro-3H-purin-6-one is used as a anti-HIV agent. It is metabolized intracellular by a series of cellular enzymes to its active moiety, dideoxyadenosine triphosphate (ddATP), which inhibits the HIV reverse transcriptase enzyme competitively by competing with natural d ATP. It also acts as a chain terminator by its incorporation into viral DNA as the lack of a 3'-OH group in the incorporated nucleoside analogue prevents the formation of the 5' to 3' phosphodiester linkage essential for DNA chain elongation, and therefore, the viral DNA growth is terminated. Solubility of the drug was found sparingly soluble in water, slightly soluble in methanol.

Figure 1: Chemical Structure of Didanosine

From the literature survey it was found that few RP-HPLC methods in pharmaceutical dosage forms and in biological samples was developed for the estimation of Didanosine^1-5. The developments of LC/MS/MS methods are also reported for the estimation of drugs^6-9. Jaiprakash N Sangshetti et al developed simple colorimetric method for its determination¹⁰. The specification and assay also are available in USP¹¹. To best of our knowledge, all the methods have been reported the retention time of more than 6 minutes. So the principle objective of this present study was, therefore, to develop a new, simple and specific HPLC method for assay of didanosine with shorter analysis time.

MATERIALS AND METHOD:

RP-HPLC chromatography was performed on a Waters -2695 separation module automatic HPLC systems with UV detector and Yl9100 HPLC system with PDA detector. An Analytical Chromosil C18 (4.6 x 150mm, 5mm) was used as a stationary phase. Afcosets digital electronics balance, Alwa pH meter were used throughout the particle. Drug sample of didanosine was kindly supplied by Pharma Traini, Hyderabad. Marketed formulation of didanosine capsules were used for the estimation of drug content. Methanol (HPLC grade, Merck), Water for HPLC (Merck), Acetonitrile (HPLC grade, Fisher Scientific), Ortho phosphoric acid, Tri ethyl amine were used.

Standard Preparation:

For the RP-HPLC method standard solution was prepared by accurately weighed and transferred 10 mg of didanosine working standard into a 10mL clean dry volumetric flask, about 7mL of diluent was added and sonicated to dissolve it completely and made volume up to the mark with the same solvent (stock solution). Further 0.3ml of above didanosine stock solution was pipetted out and transferred into a 10ml volumetric flask and diluted up to the mark with dilutent.

Test Preparation:

Sample solution was prepared by accurately weighed and transferred the capsule powder equivalent to 10 mg of didanosine into a 10mL clean dry volumetric flask. About 7mL of diluent was added and sonicated to dissolve it completely and made volume up to the mark with the same solvent (stock solution). Further 0.3ml of above didanosine stock solution was pipetted out and transferred into a 10ml volumetric flask and diluted up to the mark with dilutent.

Chromatographic Condition and Analysis of Formulation:

Chromatogram was recorded using Waters -2695 separation module, with a flow rate of 0.6 mL per min at ambient temperature and the eluent monitered at 248 nm. The separation was done on Analytical Chromosil C18 column (4.6 x 150mm, 5mm) using 200 mL of phosphate buffer pH to 3.5 adjusted with ortho - phosphoric acid (20%), 350 mL of methanol (35%) and 450 mL of acetonitrile (45%) as a mobile phase. About 20 mL of the standard and sample were injected into the chromatographic system with a run time of 6 min and the areas for the didanosine peaks were measured and calculated the % assay.

Validation:

The RP-HPLC method was validated for the parameters like linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ) and robustness as per ICH guidelines Q2(R1)¹². To determine linearity a calibration graph was obtained by plotting the graph with didanosine from the concentration range of 10 µg/mL to 50 µg/mL against peak area. Precision was determined by performing intra-day precision and inter-day precision. The standard solution was injected for five times with short interval of time (repeatability) and for five consecutive days (intermediate precision). The %RSD for the area of five replicate injections was found to be within the specified limits and results were shown in the Table 2. The accuracy of the method was assessed by determination of recovery for three concentrations (corresponding to 50, 100, 150 % of test solution concentration) covering the range of the method. For each concentration, three sets were injected and percentage recoveries were calculated (Table 3).

The robustness of the method was evaluated by assaying test solutions after slight but deliberate changes in the analytical conditions flow rate (±0.1ml/min), the proportions of organic phase in mobile phase (± 5 %, v/v) and changing the column temperature (25°C and 35°C). For each different analytical condition, the standard solution was prepared separately and analysed.

RESULTS AND DISCUSSION:

Based on interrelationship between columns, pH, mobile phase composition and its ratio the optimized parameters were set and the peak was obtained at retention time of 16.7 min (Figure 2).

The method shows good linearity in the range of 10 to 50 µg/mL. The linear regression data for the calibration plot are indicative of a good linear relationship between peak area and concentration over a wide range and the value of correlation coefficient was indicative of high significance. The data for validation parameters were tabulated in Table 1.

Table 1: Data for Linearity Study

S.N	Linearity Level	Concentration (µg/mL)	Peak Area
1		10	1057205
2		20	1848398
3		30	2603891
4		40	3433898
5		50	4106800
Correlation Coefficient			0.999

Table 2: Results for Precision Study

S.N	Peak Area
S.N	Intra-day Precision	Inter-day Precision
1	2608834	2592793
2	2608175	2585469
3	2613654	2601879
4	2621386	2598410
5	2632749	2608297
Average	2616959	2597370
Standard Deviation	10283.8	8709.1
%RSD	0.39	0.34

Table 4: Robustness Study Data

Parameters		USP tailing factor	USP Plate Count	% RSD
Flow Rate (mL/min)	0.5	1.48	2553	0.6
	0.6	1.48	2679	0.2
	0.7	1.48	1899	0.1
Organic Phase Composition (%)	5% less	1.57	2215	0.1
	Actual	1.48	1679	0.3
	5% more	1.69	1911	0.2
Column Temp. (°C)	25	1.56	2245	0.3
	30	1.69	2256	0.2
	35	1.57	1968	0.1

The results of % relative standard deviation (%RSD) of the peak areas of five replicates for repeatability study and intermediate precision were found to be 0.39 % and 0.34 % respectively (Table 2). The accuracy of the method was determined by performing the recovery experiment at three levels (50%-150%). The % recovery obtained between 99.6– 101.5% proved that the method was accurate (Table 3).

There was no significant change in the system suitability factors of cefoxitin sodium when the organic composition, flow rate and column temperature were changed. The low values of the %RSD indicated that the method was robust enough and the results were tabulated in Table 4.

From the stability study, difference in % assay for drug product with respect to initial was NMT 3.0 %. It was established that the mobile phases, test and standard solutions were stable for 1 day on bench top and stable for 2 days in refrigerator.

Figure 2: Chromatogram for Didanosine Standard

CONCLUSION:

A simple, rapid and reproducible RP-HPLC method was developed and validated for the determination of didanosine. The method was validated for system suitability, precision, accuracy, linearity, and robustness. The system suitability parameters were within limit, hence it was concluded that the system was suitable to perform the assay. Correlation coefficient of 0.9991 it was closer to one, it showing good correlation between peak area and concentration of the drug solution. The method was robustness as observed from insignificant variation in the results of analysis by changes in organic modifier, flow rate and temperature separately and analysis being performed, on different systems. The developed isocratic LC method offers simplicity, selectivity, precision and accuracy. In the proposed method symmetrical peaks with good resolution were obtained. The RP-HPLC method was sensitive and precise. However, this method can be used for the routine analysis of the estimation of didanosine in pharmaceutical dosage form.

REFERENCES:

1. Sathiya Sundar R, Carolin Nimila1 I, Ashok Kumar J, Vijaya Kumar TM, Poovi G and Sankar Anand R. Analytical method development and validation of different marketed didanosine tablets by RP-HPLC, International Journal of Research and Pharmaceutical Science. 1(2); 2010: 205-208.

2. Antonia Maria Cavalcanti de Oliveira, Teresa Cristina Raposo L¨owena, L´ucio Mendes Cabral, Elizabeth Moreira dos Santos, Carlos Rangel Rodrigues, Helena C. Castro and Tereza Cristina dos Santos, Development and validation of a HPLC-UV method for the determination in didanosine tablets. Journal of Pharmaceutical and Biomedical Analysis. 38(4); 2005: 751–756.

3. Naser L Rezk, Richard R Tidwell and Angela DM Kashuba. Simultaneous determination of six HIV nucleoside analogue reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance detection. Journal of Chromatography B: Analytical technologies in the biomedical and life sciences. 791(1-2); 2003: 137–147.

4. Stefania Notari, Alessio Bocedi, Giuseppe Ippolito, Pasquale Narciso, Leopoldo Paolo Pucillo, Gianna Tossini, Raffaele Perrone Donnorso, Francesco Gasparrini and Paolo Ascenzial. Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography. Journal of Chromatography B: Analytical technologies in the biomedical and life sciences. 831(1-2); 2006: 258–266.

5. Maria Lluisa Rosell-Rovira, Leonor Pou-Clave, Rosa Lo-pez-Galera and Caries Pascual-Mostaza. Determination of free serum didanosine by ultrafiltration and high-performance liquid chromatography. Journal of Chromatography B: Biomedical Applications. 675(1); 1996: 89-92

6. Vincent Bezya, Philippe Morin, Philippe Couerbe , Ghis-laine Leleu and Luigi Agrofoglio, Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer. Test of this new volatile medium-pH for HPLC–ESI-MS/MS. Journal of Chromatography B: Analytical technologies in the biomedical and life sciences. 821(2); 2005: 132–143.

7. Xavier Cahours, Thanh Thu Tran, Nathalie Mesplet, Claudine Kieda, Philippe Morin and Luigi Agrofoglio. Analysis of intracellular didanosine triphosphate at sub-ppb level using LC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis. 26(5-6); 2001: 819–827

8. Stefania Notari, Carmine Manconea, Tonino Alonzi, Marco Tripodi, Pasquale Narciso and Paolo Ascenzia. Determination of abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine concentration in human plasma by MALDI-TOF/TOF. Journal of Chromatography B: Analytical technologies in the biomedical and life sciences. 863(2); 2008: 249–257.

9. Yong Huanga, Elisabeth Zurlinden, Emil Lin, Xiaohua Li, Jason Tokumoto, Jeff Golden, Andrew Murre, John Engstrom and John Conte Jr. Liquid chromatographic–tandem mass spectrometric assay for the simultane-ous determination of didanosine and stavudine in human plasma, broncho alveolar lavage fluid, alveo-lar cells, peripheral blood mononuclear cells, seminal plasma, cerebrospinal fluid and tonsil tissue. Journal of Chromatography B: Analytical technologies in the biomedical and life sciences. 799(1); 2004: 51–61Jaiprakash N Sangshetti, Parag A Kulkarni and Devanand B Shinde. Spectrophotometric determination of didanosine in bulk and tablet formulation. Trends in Applied Sciences Research. 2; 2007: 71-75.

Received on 19.11.2012 Accepted on 22.11.2012

Asian J. Pharm. Ana. 2(4): Oct. - Dec. 2012; Page 118-121

% Level	Amount of Std Drug Added (mg)	Amount Found (mg)	Peak Area	% Recovery	Mean Recovery
50 %	15.0	15.22	3749470	101.45 %	100.27 %
100 %	20.0	19.93	4910900	99.65 %
150 %	25.0	24.92	6141156	99.69 %