mation of doxofylline in pharmaceutical formulations. The present work describes a simple, precise and accu rate RP HPLC method for estimation of doxofylline.
ISSN 10619348, Journal of Analytical Chemistry, 2010, Vol. 65, No. 3, pp. 293–297. © Pleiades Publishing, Ltd., 2010.
ARTICLES
Development and Validation of Rapid HPLC Method for Determination of Doxofylline in Bulk Drug and Pharmaceutical Dosage Forms1 Ashu Mittal and Shikha Parmar ITS—Paramedical (Pharmacy) College, Department of Pharmaceutics, Muradnagar (Ghaziabad), India Received May 19, 2008; in final form, June 25, 2009
Abstract—A simple, selective, rapid, and economical reversed phase high performance liquid chromatogra phy(RPHPLC) method for the determination of doxofylline in the commercial dosage form has been devel oped and validated. The separation and quantification were achieved on an HiQ Sil C 18 W column using a mobile phase of acetonitrile : buffer (50 : 50), pH 3, at a flow rate of 1 mL/min with detection of analyte at 272 nm. The separation was achieved within 3.1 ± 0.3 min for doxofylline sample. The method showed good linearity in the range of 10–80 µg/mL. The intra and inter day RSD ranged from 0.37–0.53%. The recovery (mean ± S.D.) of low, middle and high concentrations were 100.04 ± 0.80, 100.01 ± 0.20, 100.07 ± 0.30 respectively. Limit of detection and limit of quantification were 0.03 and 0.1 µg/mL, respectively. DOI: 10.1134/S1061934810030147 1
Theophylline (1,3dimethylxanthine), especially in sustained release preparations, is widely prescribed in the therapy of asthma [1]. Among the theophylline analogs recently introduced for such therapy is dox ofylline (2[7'theophylline methyl]1,3dioxolane), which is claimed to retain the therapeutic properties of theophylline but have a lower incidence of side effects [2, 3]. It is given by mouth in doses of up to 1200 mg daily [4]. Although various bioanalytical methods for estimation of doxofylline in human serum [5–8] and spectrophotometric method for estimation of doxofyl line in dosage form [9] have been reported in the liter ature, there is no HPLC method reported for the esti mation of doxofylline in pharmaceutical formulations. The present work describes a simple, precise and accu rate RPHPLC method for estimation of doxofylline in commercial dosage form. The results of analysis were validated using International Conference on Harmonization (ICH) guidelines [10]. EXPERIMENTAL Reagents. Acetonitrile (HPLC grade) was pro cured from Qualigens, India; MilliQwater was pur chased from Rankem, India. Orthophosphoric acid was obtained from E. Merck, India. Reference stan dards of doxofylline were procured from Zydus Cadila, Ahmedabad, India. Instrumentation. The HPLC system used in the study was Jasco (Japan), a rheodyne injection valve equipped with a 20 µL loop (Rheodyne, USA), a dec tector (Jasco, UV2075 plus intelligent dectector 1 The article is published in the original.
UV/Vis), LCNET II/ADC integrator was used. Sep aration was accomplished on a Hiq SilC 18 W col umn. The mobile phase was composed of aetonitrile : buffer (50 : 50 v/v), pH adjusted to 3.0 ± 0.2 using orthophosphoric acid at a flow rate 1 mL/min. The mobile phase was filtered through 0.45 μm Millipore HVLP filter and degassed by sonication before use. The UV detection was set at 272 nm. The retention time was 3.1 ± 0.3 min (Fig. 1). The validation of the proposed method was also carried out according to ICH guidelines. Method development. To optimize the chromato graphic conditions, the effect of chromatographic variables such as mobile phase, pH, flow rate and sol vent ratio were studied. Various solvent systems were tried for the development of a suitable HPLC method for determination of Doxofylline in bulk drug and pharmaceutical formulations. Mobile phase tried for this purpose were acetonitrile : buffer (80 : 20), aceto nitile : buffer (50 : 50), methanol : water (70 : 30), methanol : water (50 : 50), methanol : water:aceto nitile (35 : 30 : 35). The condition that gave the best resolution and symmetry was selected. Same solvent system was used for the extraction of the drug from the formulation containing excipients which was used for quantification. Calibration Curve. A stock solution of doxofylline (100 µg/mL) was prepared by dissolving 50 mg of drug in 100 mL of mobile phase, further 2 mL of this solu tion were transferred to a 10 mL volumetric flask and volume was made up to 10 mL to obtain 100 µg/mL solution. Different concentrations (1–80 µg/mL) were made for the preparation of calibration curve from the stock solution. The mobile phase after filtra
293
294
ASHU MITTAL, SHIKHA PARMAR µV 2.5E + 05 2.0E + 05 1.5E + 05 1.0E + 05 5.0E + 04 0.0E + 00 1.00
2.00
3.00
4.00 min
Fig. 1. UV spectrum of doxofylline.
tion through a 0.45 μm membrane filter was delivered at 1.0 mL/min for column standardization, and base line was continuously monitored during the process. The UV scan of doxofylline was performed between 200–400 nm and wavelength of detection was selected at 272 nm. The doxofylline UV spectrum is shown in Fig. 2. The prepared dilutions were injected serially and areas under the peaks were calculated for each dilution. The stability of drug in solution during anal ysis was determined by repeated analysis of samples during the course of experimentation on the same day and also after 48 h storage of drug solution at labora tory bench conditions and in the refrigerator. Method Validation. Linearity. The concentrations of doxofylline within 0–80 µg/mL were prepared from stock solution (100 µg/mL) and areas under peak were calculated. The graph was plotted between concentra tion and area under peak for linearity. Precision. Precision was considered at two levels, i.e., repeatability and intermediate precision. Repeat ability of sample application was determined as intra day variation whereas intermediate precision was determined by carrying out interday variation for the determination of doxofylline at three different con centration levels of 8, 6, and 32 µg/mL. Spectrum 2.00 A
272.0 nm
1.161 A
(0.500/div)
–0.10 A 200.0 nm
(50/div)
400.0 nm
Fig. 2. Typical HPLC chromatogram of doxofylline.
Accuracy as Recovery. Accuracy of the method was studied by recovery experiments. About 5.5 mg of pla cebo and 16, 32, and 48 mg of doxofylline were trans ferred into a 100 mL volumetric flask. About 50 mL of mobile phase were added, sonicated for 10 min and shaken for 5 min. The volume was made up to the mark with mobile phase and mixed. Solution was fil tered through a 0.45 µm Millipore HVLP filter and the filtrate collected by discarding few mL of filtrate. Finally 5.0 mL of this solution were diluted to 50.0 mL with mobile phase. The final concentrations for 50, 100, and 150% accuracy were 16, 32, and 48 µg/mL of doxofylline, respectively. Specificity. A synthetic mixture containing 16 mg of doxofylline and 30 mg each of starch, lactose, magne sium stearate, and avicel, which are present as excipi ents in the tablet dosage form, was accurately weighed and transferred to a 50 mL volumetric flask. The mix ture was shaken well with 30 mL of methanol and then diluted to volume with methanol. After filtration, 5 mL of the filtrate were transferred to a 50 mL volu metric flask and diluted to volume with mobile phase, to furnish a final solution containing 32 µg/mL of dox ofylline. Robustness. Robustness was carried out to evaluate the influence of small but deliberate variations in the chromatographic conditions for the determination of doxofylline. Robustness of the method was deter mined by changing the flow rate (0.8 and 1.2 mL/min), mobile phase ratio (±10%), pH (±10%), and temperature (±10%). Assay of Commercial Dosage Form. Accurately weighed quantity of tablets powder equivalent to about 80 mg of doxofylline in to a 250 mL volumetric flask was transferred. About 100 mL of mobile phase was added, the solution was sonicated for 20 min with con tinuous shaking at 30°C. Volume was made up with mobile phase. The solution was filtered through a 0.45 µm HVLP filter paper by discarding first few mL of the filtrate. A 5.0 mL aliquot of the solution was diluted to 50 mL with mobile phase to make final con centration 32 μg/mL of doxofylline. Standard solu
JOURNAL OF ANALYTICAL CHEMISTRY
Vol. 65
No. 3
2010
DEVELOPMENT AND VALIDATION OF RAPID HPLC METHOD
pose were acetonitrile : buffer (80 : 20), acetonitrile : buffer (50 : 50), methanol : water (70 : 30), methanol : water (50 : 50), methanol : water : acetonitrile (35 : 30 : 35). Internal standard was not used as there was no extrac tion or separation step involved. The chromatogram obtained with acetonitrile : buffer (50 : 50) solvent sys tem was found to have very good symmetry (1.4 ± 0.04), with lowest Rt (3.07 ± 0.03) and sharp well defined peak. Therefore the mixture of acetoni trile:buffer (50 : 50), pH 3.0 was chosen as mobile phase. The drug was stable for a period of 48 h at labo ratory temperature and under refrigerator temperature in acetonitrile : buffer (50 : 50) mixture. Method Validation. Linearity. The linearity range of doxofylline solutions was obtained as 10–80 µg/mL. The linear regression equation was y = 15175x – 31.407 with regression coefficient of 0.999 (Table 1). Precision. Results of repeatability and intermediate precision were expressed in terms of % RSD and are shown in Table 2. The low value of RSD indicates good repeatability of the proposed method. Accuracy as Recovery. The values of drug recovered, mean recovery and % RSD are shown in Table 3, which indicate satisfactory accuracy of the proposed method. Specificity. The specificity of the method was tested by calculating the percentage recovery of each compo nent in the presence of possible interfering materials such as starch, lactose, magnesium stearate, and avicel. The results are presented in Table 4, which shows that separation of analytes from the excipents was complete. Robustness of the method. There was no significant change in the retention time of doxofylline by chang
Table 1. The results of linear regression for doxofylline Parameter
Value
Concentration range, µg/mL
10–80
Slope
1.52 × 104
Intercept
–3.1 × 101
Correlation coefficient
0.9999
Table 2. Intraday and interday precision of the method (n = 5) Concentration, µg/mL
Intraday precision Interday precision Mean
RSD, %
Mean
RSD, %
8.0
8.03
1.00
7.98
1.50
16.0
16.12
1.12
16.09
1.37
32.0
31.92
0.47
32.08
0.97
295
tions in the concentration range of about 32 µg/mL were also prepared. RESULT AND DISCUSSION Method development. The proposed HPLC proce dure was optimized with a view to develop a suitable analytical method. Mobile phases tried for this pur
Table 3. Recovery values obtained for the determination of doxofylline Drug recovered Recovery level, %
50
100
150
Set
Drug added, mg mg
%
1
16.01
16.09
99.50
2
15.96
15.79
101.07
3
16.00
16.07
99.56
1
32.02
32.76
97.74
2
32.05
32.00
100.06
3
32.00
31.30
102.24
1
47.98
46.31
103.61
2
48.00
48.11
99.77
3
48.03
49.59
96.85
JOURNAL OF ANALYTICAL CHEMISTRY
Vol. 65
No. 3
2010
Mean recovery
RSD, %
100.04
0.80
100.01
0.20
100.07
0.30
296
ASHU MITTAL, SHIKHA PARMAR
Table 4. Specificity of method* Added, µg/mL
Recovered, µg/mL
Recovery, %
32.00
32.32
101.0
32.00
32.61
101.91
32.00
31.7
99.22
32.00
32.13
100.44
Mean
100.44
RSD
1.12
Application of method to assay doxofylline in tablet. The method was used for the determination of dox ofylline in tablet formulations. The result obtained (Table 6) showed that percentage recoveries were high and RSD values were low, which confirms the method is suitable for routine determination of doxofylline in its pharmaceutical preparation. Figure 1 show a typi cal chromatogram obtained from analysis of a tablet formulation. Stability. The stability of doxofylline in solution was checked by determining the percentage deviation of the amounts present in solution after 48 h at room temperature in comparison with the amount at zero time. The results obtained after 48 h showed no signif icant variation; the percentage deviation was less than 2% of the initial amount. This is indicative of good sta bility of each component in the mixture over a period of 48 h.
* Each sample contained 60 µg/mL each of starch, lactose, magne sium stearate and avicel.
CONCLUSIONS ing the flow rate, pH, mobile phase ratio and temper ature. Low value of % RSD indicates the robustness of the method as shown in Table 5. Limit of detection (LOD) and quaLimit of detection (LOD) and quantification (LOQ)ntification (LOQ). The LOD and LOQ were calculated based on the concen tration of solute for which the peak heights were 3 times the baseline noise. LOD & LOQ were 0.03 μg/mL and 0.1 µg/mL, respectively. These low values were most probable due to the smooth baseline.
The HPLC method proposed in this study using common reagents and simple sample preparation pro cedures is particularly appropriate for the routine analysis of doxofylline in tablet dosage form. This method has the advantages of simplicity, precision, accuracy, sensitivity and quantification of doxofylline compared with other reported methods and can be employed for its assay in dosage form with a single injection. The selectivity of the chosen chromato graphic systems was ascertained. Excipients showed no peak in the range of the retention times corre sponding to the analyte.
Table 5. Robustness of the method (n = 5) Changed parameters Temperature
pH
Flow Rate
Mobile phase ratio
RSD, % Normal conditions
–5°C
+5°C
0.63
0.22
1.77
Normal conditions
–0.2 units
+0.2 units
0.63
0.14
0.11
Normal conditions
–10%
+10%
0.63
0.59
0.31
Normal conditions
–2%
+2%
0.63
0.07
0.16
JOURNAL OF ANALYTICAL CHEMISTRY
Vol. 65
No. 3
2010
DEVELOPMENT AND VALIDATION OF RAPID HPLC METHOD Table 6. Results of assay of doxofylline (µg/mL) in commercial dosage form (n = 3) Added
Recovered
Recovery, %
32.00 32.00 32.00 Mean RSD
32.8 32.56 32.48
100.56 101.75 101.50 101.27 0.62
ACKNOWLEDGMENTS This work was supported by grants from the Uttar Pradesh Technical University, Lucknow. We thank Zydys Cadila (Ahmedabad, India) for the gift sample of doxofylline. REFERENCES 1. Uden, D.L., Wysttt, R.A., and Zaske, D.E., Post Grad. Med., 1964, vol. 75, p. 247.
JOURNAL OF ANALYTICAL CHEMISTRY
Vol. 65
297
2. Simona, F.E.R., Simona, K.J., and Bierman, C.W., J. Allergy Clin. Immunol., 1975, vol. 56, p. 347. 3. Grossi, E., Biffignandi, P., and Franzaone, J.S., Eur. Rev. Med. Pharmacol. Sci., 1988, vol. 10, p. 1. 4. Bologna, E., J. Int. Med. Res., 1990, vol. 18, p. 282. 5. Lagana, A., Bizzarri, M., Marino, A., and Mancini, M., Biomed. Chromatogr., 1990, vol. 4, p. 205. 6. Tagliaro, F., Dorizzi, R., Frigerio, A., and Marigo, M., Clin. Chem., 1990, vol. 36, p. 113. 7. Sreenivas, N., Narasu, M.L., Shankar, B.P., and Mul langi, R., Biomed. Chromatogr., 2008, vol. 22, p. 654. 8. Gannu, R., Bandari, S., Sudke, S.G., Roa, Y.M., and Shankar, B.P., Acta Chromatogr., 2007, vol. 19, p. 149. 9. Kamila, M.M., Mondal, N., and Ghosh, L.K., Ind. J. Chem. Tech., 2007, vol. 14, p. 523. 10. International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuti cals for Human Use: Harmonized Triplicate Guidelines on Validation of Analytical Procedure: Methodology Rec ommended for Adoption at Step 4 of the ICH Process on November 1996 by ICH Steering Committee, Switzer land: IFPMA.
No. 3
2010
