Validation of analytical method by HPLC for determination of dapsone in polymeric nanocapsules based on crude rice brain oil

Article history: Received on: 08/03/2017 Accepted on: 30/04/2017 Available online: 30/07/2017 Dapsone is a sulfone used for the treatment of skin diseases such as psoriasis. Its clinical use by the oral route is limited. For counteract this hindrance, innovative systems based on polymeric nanocapsules containing rice bran oil and dapsone have been developed. Thus, an analytical method using HPLC for quantification of dapsone in polymeric nanocapsules based on rice brain oil was developed and validated. The separation was achieved on a C18 reverse phase column (Phenomenex, 4.6 × 150 mm, 5 μm) using a mobile phase composed of acetonitrile– 1.5 % acetic acid (25:75 v/v) at a flow rate of 0.7 mL.min −1 . The injection volume was 20 μL and the wavelength of detection was 230 nm. The method was linear in the range of 5-25 μg.mL −1 , with a correlation coefficient of 0.9999. Precision and accuracy analysis showed low relative standard deviation (lower than 2) and recovery percentage in the range of 99.72 – 106.25%. The limits of detection and quantification were 0.41 and 1.24 μg.mL −1 , respectively. The analytical method was specific, linear, precise and exact, showing that this procedure can be applied in quantification of dapsone-loaded polymeric nanocapsules based on rice brain oil.


INTRODUCTION
Dapsone (4,4'diaminodiphenylsulfone, C 12 H 12 N 2 O 2 S) (Figure 1), molecular weight of 248.31 g.mol-1 , is a white, crystalline and odorless powder.It is sparingly soluble in water, easily soluble in acetone and lightly soluble in ethanol and methanol (Wozel et al., 2010).activity.Dapsone is also used for other chronic skin diseases such as psoriasis that is a chronic inflammatory disease that has a predilection for skin and joints (Zhu and Stiller, 2001).When used orally, dapsone presents some undesirable effects such as hemolytic anemia and methemoglobinemia (Wozel et al., 2010).Due to the high incidence of side effects, the development of a topical formulation of the drug becomes interesting, however, the clinical efficacy of a drug, including dapsone, administered topically is limited since the most of drugs have unsuitable physico-chemical characteristics for overcoming the skin barriers, in particular the stratum corneum (Schafer-Korting et al., 2007).Thus, the development of carrier systems in order to improving the therapeutic effect of drugs administered by the topical route is an interesting alternative.Among these systems, the polymeric nanoparticles are noteworthy.Polymeric nanoparticles are vesicular or matricial colloids based on polymers.These vesicular carriers constituted of an oil core surrounded by a polymeric wall are named nanocapsules (Bernardi et al., 2011).
In this work, dapsone loaded-nanocapsules were developed using crude rice bran oil as the oily core and poly (εcaprolactone) as the polymer.Unsaponifiable matter of crude rice bran oil contains high levels of components with antioxidant properties, such as tocopherols, tocotrienols and gamma-oryzanol.Besides, the crude rice bran oil presents moisturizing, humectants and UV protection properties.Thus, it is a multifunctional component for the formulation of cosmetic and dermatological products (Juliano et al., 2005), including the innovative dapsoneloaded polymeric nanocapsules intended for the treatment of psoriasis.Several methods for the quantification of dapsone have been related in the literature.These studies were focused on identification of degradation products (Bardsley et al., 2011), evaluation of stability (Nahata et al., 2000), quantification of dapsone and its metabolites in muscle, plasma (Kwadijk and Torano, 2002) and urine (Abuirjeie et al., 1991) in biological evaluation of the drug in humans or animals (Hela et al., 2003).Apart of these, a method for quantification of dapsone in theospheres, a special lipid nanoparticulated system, has been developed (Santos et al., 2012).The drug extraction from nanoparticles for quantification purposes is extremely difficult, and it requires the use of different solvents that allow the disruption of the carrier and the extraction of the nanoencapsulated substance, take into account the specific characteristics of the carrier system.Therefore, the development of a simple, fast and accurate method for the extraction and quantification of dapsone from this particular kind of nanocapsules, based on rice bran oil is highly desirable due to complexity of this system that include polymer, surfactants and a vegetal component.Thus, this study aimed to develop and validate an analytical method for quantification of dapsone in polymeric nanocapsules based on crude rice brain oil by high performance liquid chromatography (HPLC).

Chromatographic equipment and conditions
The analyses were carried out on a Proeminence ® (Shimadzu) high performance liquid chromatograph equipped with binary pump and diode-array detector.
The separation was achieved on a C18 reverse phase column (Phenomenex, 4.6 × 150 mm, 5 μm) using a mobile phase composed of acetonitrile-1.5 % acetic acid (25:75 v/v) at a flow rate of 0.7 mL.min −1 .The injection volume was 20 μL and the wavelength of detection was 230 nm.

Preparation and characterization of dapsone-loaded nanocapsules
The nanocapsules were prepared by interfacial deposition method of the preformed polymer (Jager et al., 2009).The organic phase, consisting of acetone (30 mL), dapsone (0.01 g), PCL (0.1 g), sorbitan monostearate (0.077 g) and crude rice bran oil (0.15 g), was poured into the aqueous phase (50 mL) containing polysorbate 80 (0.077 g), under moderate magnetic stirring.After 10 min acetone was eliminated and the suspension was concentrated under reduced pressure.Empty nanocapsules (without dapsone) were prepared and used as a control.
The nanoparticles were characterized in terms of mean diameter and size distribution by laser diffractometry (Mastersizer ® 2000, Malvern Instruments).In the drug content evaluation, stock solution of dapsone (1000 μg.mL -1 ) was prepared by dissolving accurately weighed 10 mg of the drug in acetonitrile using 10 mL volumetric flask.Standard solution was prepared by dilution of the diluted stock solution with mobile phase to obtain solutions in a final concentration of 15 μg.mL -1 .To prepare the sample solutions, 0.15 mL of nanocapsule suspension (1 mg.mL -1 of dapsone) was transferred into a 10 mL volumetric flask, with 10 mL of acetonitrile, obtaining the final concentration of 15 µg.mL -1 .This flask was kept in an ultrasonic bath for 30 min, causing partial dissolution of the polymer and release of the active contained into the nanostructure.The solutions were filtered through a 0.45 µm membrane filter and analyzed by HPLC, determining the total concentration of dapsone.Free drug was also determined by HPLC in the ultrafiltrate after the separation of the nanoparticles by an ultrafiltration-centrifugation technique (Ultrafree-MC ® 10,000MW, Millipore), at 12000 rpm for 5 min.In this case, the ultrafiltrate was directly injected in HPLC.Encapsulation efficiency (%) was calculated by the difference between the total and free drug concentrations.In addition, the pH value of the nanocapsule suspension was determined.

Validation of analytical method
The validation of the analytical method was performed according to the International Conference on Harmonization Guidelines (ICH) (ICH, 2005) by validation of analytical procedures.The parameters evaluated in the development of this work were: specificity, linearity, intermediate precision, repeatability, accuracy, detection and quantification limits.The specificity of the method was evaluated through comparative analyzes of the suspensions of empty nanocapsules (without dapsone) and the suspensions containing the drug.
To study the linearity,calibration curves (n=3) (area as a function of dapsone concentration) were obtained.Stock solution of dapsone (1000 μg.mL -1 ) was prepared by dissolving accurately weighed 10 mg of the drug in acetonitrile using 10 mL volumetric flask.Samples solution was prepared by dilution of the diluted stock solution with mobile phase to obtain solutions in a final concentration 5, 10, 15, 20 and 25 μg.mL -1 .Precision was assessed by the analysis of dapsone-loaded nanocapsule suspension (n=6) in one single day (repeatability) and through analysis of samples in three different days (intermediate precision).For this, 0.15 mL of nanocapsule suspension (1 mg.mL -1 of dapsone) was transferred into a 10 mL volumetric flask, with 10 mL of acetonitrile, obtaining the final concentration of 15 µg.mL -1 .This flask was kept in an ultrasonic bath for 30 min.The samples were filtered through a 0.45 µm membrane filter and analyzed by HPLC.The accuracy was determined in three different concentrations (low, intermediate and high).A standard solution of dapsone was prepared (1000 mg.mL -1 ) and aliquots of 50 μL, 150 μL, and 250 μL of this solution were transferred to a 10 mL volumetric flask containing 100 μL of empty nanocapsule suspension (without drug) and acetonitrile was used to reach the final volume.Thus, the evaluated concentrations were 5 μg.mL -1 , 15 μg.ml -1 and 25 μgml -1 performed in triplicate.The limits of detection (LOD) and quantitation (LOQ) were calculated using the calibration line directly.The factors (3.3 and 10, respectively) were multiplied by the ratio from the residual standard deviation and the slope of the standard curve, according to the guidelines (ICH, 2005).

RESULTS AND DISCUSSION
The dapsone-loaded nanocapsules prepared using crude rice bran oil presented aspect similar to a milky bluish opalescent fluid.The empty nanocapsules presented mean diameter of 167 0.1 nm and the presence of dapsone did not change the particle size which was 168 0.1 nm.Size distributions showed monomodal profiles with Span values of 1.66 and 1.69 for empty and dapsone-loaded nanocapsules, indicating narrow size distributions for both suspensions.The pH values of the suspensions were close to 6.0.The dapsone-loaded nanocapsule presented drug content close to their theoretical value (1 mg.mL −1 ) and the drug encapsulation efficiency was 83.2%.
Regarding the validation of analytical method, Figure 2 shows the chromatogram of the nanocapsules suspensions of dapsone and the chromatogram of the suspensions without the drug.Prior the analysis, the sample of empty nano capsule was processed in the same manner as nanocapsules containing dapsone, using the same solvent (acetonitrile).As shown in the Figure 2, there was no interference in the chromatogram for the evaluation of the drug in the nanocapsule formulations.Thus, the analytical method presented is specific to quantify dapsone in nanocapsule based on rice brain oil, which is consistent with the official specifications.In linearity assay, the straight line equation (y = 106073.99x+ 40990.53)was obtained by linear regression studies between the concentration of dapsone and their respective responses, yielding a coefficient of determination of 0.9999, which is characterized linear and suitable for such use over the 5-25 μg.mL -1 range.The slope and intercept (± SD, n = 3) were 40990.53± 312.04 and 106073.99± 6007.53, respectively.The ANOVA results showed that the regression equation was linear (F calculated = 1.4.10 4 >F critical = 4.96; P = 0.05) with no deviation from linearity (F calculated = 2.05 <F critical = 3.71; P = 0.05).The values obtained for the LOD and LOQ were 0.41 and 1.24 μg.mL -1 , respectively, indicating a good sensitivity of the method for determining of dapsone.From the results obtained in the development of the method, it is concluded that the calibration curve can be used for quantification of experimental values of dapsone, whereas the determination coefficient was higher than 0.99, confirming the quality of the obtained calibration curve (ICH, 2005).In the performed intra-day tests the values were between 75.02 and 78.18% with RSD of 1.68 on the first day of the analysis.Already on the second day of the test, recovery values were between 77.25 and 78.74% with RSD of 1.00.Finally, on the third day of the test, the values were between 77.73 and 79.95% with RSD of 1.54.Thus, the parameter has been confirmed by accurate alignment of the different results obtained for the same sample.Regarding the accuracy test, the results obtained are shown in Table 1.Values of recovery percentages were satisfactory, since it remained between 99.72 and 106.25%.Thus, the method can be considered accurate for quantification of dapsone in polymeric nanocapsules based on crude rice brain oil.

Fig. 2 :
Fig. 2: Comparison of chromatograms obtained from the analysis of the dapsone-loaded nanocapsules (A) and empty nanocapules (B).

Table 1 :
Experimental values of accuracy of the method for dapsone quantification in polymeric nanocapsules.