International Journal of Drug Development and Research

Keywords

Response surface methodology, Central composite design, Derringer’s Desirability function, RP-HPLC, Dutasteride, Tamsulosin HCL.

Introduction

Dutasteride (DUT) is 4- azasteroid compound, chemically it is 5 , 17 -N-{2, 5 bis(trifluoromethyl)phenyl}-3-oxo-4-azaandrost-1- ene-17-carboxamide(Figure 1). It is a selective inhibitor of both the type 1 and type 2 isoforms of steroid 5 alpha-reductase, an intracellular enzyme that converts testosterone to dihydrotestosterone. It is used in the treatment of benign prostatic hyperplasia. [1 – 3]

Tamsulosin HCl (TAM) is a selective antagonist at alpha-1A and alpha-1B-adrenoceptors in the prostate, prostatic, urethra, and bladder neck. Blockage of these receptors causes relaxation of smooth muscles in the bladder neck and prostate, and thus decreases urinary outflow resistance in men. It is chemically (-)-(R)-5-[2-[[2-(O-ethoxyphenoxy) ethyl] amino] propyl]-2- methoxybenzenesulfonamide, monohydrochloride (Figure 2) [1,2,4].

Literature survey revealed that, few analytical methods such as, HPLC [5,6], HPTLC [7], UV [8-10], Capillary electrophoresis [11] and LC –MS [12] were reported for the estimation of DUT and TAM either individually or combined with other drugs. There is no article available in the literature concerning chemometrics approach used for the method development for the simultaneous estimation of DUT and TAM. Developing and optimizing isocratic HPLC methods [13] are a difficult procedure that requires instantaneous determination of several factors. In order to optimize more than one response at a time, the chemometric methods which includes factorial design 14 and response surface methodology [15 – 19] were applied.

Experimental:

Samples: Analytical pure samples of DUT (99.81%) and TAM (99.72%) were kindly donated by AN therapeutics, (Pondicherry, India). HPLC grade methanol (S.D fine chemical Ltd., Mumbai, India) and HPLC grade water, arranged from Milli-QAcademic system, Millipore, Bangalore, India, were used throughout the experiments. The pharmaceutical formulation used in this study was URIMAX-D tablets(Cipla Private Ltd, Mumbai, India) procured from the local market and labelled to contain 0.5mg DUT and 0.4mg TAM per tablet.

Instrumentation and Chromatographic conditions:

A shimadzu HPLC system consist of LC-10AT-vp Solvent develivery system (pump), SPDM – 10AVP photodiode array detector, Rheodyne injector with 20μL loop volume, LC- Solution assisted for data collections and processing. The mobile phase consisted of methanol and water(80:20) and the flow rate was 1.2ml/min. Separation was achieved using a 150mm X 4.6 mm (i.d.) Phenomenex luna C18 column with an average particle size of 5μ and the column was kept at an ambient temperature. The column effluent was monitered at 235 nm by PDA detection. The mobile phase was filtered through 0.45μ filter before using.

Standard stock solution: Standard stock solutions of 100 μg/ml of DUT and TAM were prepared separately in methanol. From the stock solutions, the mixed standard solutions were prepared to contain 30 μg/ml of DUT and 24 μg/ml TAM.

Sample solution: Twenty tablets were accurately weighed and finely powdered. A quantity of powder weight equvalent to 1.5 mg of DUT and 1.2 mg of TAM were weighed and transferred to a 50 ml volumetric flask. Resulting solution was sonicated for 15 minutes. Then the final volume was adjusted with methanol and filtered by vaccum filtration. The filtrate was centrifuged at 10,000 RPM for 30 minutes. The clear supernatent solution was transferred to a separate flask without disturbing the sediment.

Software: Experimental design, data analysis and desirability function calculations were performed by using Design Expert® trail version 7.0.0.(Stat- Ease Inc., Minneapolis)

Experimental design: In RSM the most popular design CCD was selected for this experiment. Two factors at two levels was used to optimize the chormatographic conditions. Percentage of methanol in the mobile phase (A) and flow rate (B) was selected in the variation of levels of 70-80 % v/v and 0.8ml/min – 1.2 ml/min respectively. Two responses were measured in each run, of total 13 runs. Retention time (tR2) and resolution (Rs2) responses were measured. CCD’s are intended to assess the coefficient of quardartic model, consist of 4 factorial, 4 axial and 5 center points.

Results and Discussion:

Optimization of design: Factorial design and RSM are usually included for the optimization of isocratic HPLC conditions in chemometric methods. Choice of key factors examined for optimization were based on initial experiments and from the literature. The two factors (A and B) and two responses (tR2 and Rs2) were selected for the optimization process. Totally 13 runs were generated by the software and all the experiments were performed in randomized order to decrease the effects of uncontrolled variables that may bring in unfairness on the measurements. The design and measured responses are represented in Table 1.

Before initialising an optimization procedure, it is obligatory to investigate the curvature term using CCD with center points. ANOVA generated for CCD publicized that, curvature is important for both the responses (tR2 and Rs2). Since p-value is less than 0.05, quadratic model was considered. The quadratic mathematical model for three independent factors was given in Equation (1).

Statistical parameters obtained from ANOVA for the reduced model were given in Table 2. In order to get more realistic model, unimportant terms with corresponding p value > 0.05 were removed during backward elimination process. Since R2 always decreases when a regressor variable is eliminated from regression model, the adjusted R2 which takes the number of regressor variables in to account is generally selected in statistical modeling [20].

The adjusted R2 values were well with in the satisfactory limits of R2 > 0.88 [21], publicized that the experimental datas are in good fit with the second order polynomial eauations. Since p value is < 0.05, reveals that, all the reduced models are significant. In this study, the signal (response) to noise (deviation) ratio was found to be in the range of 54 - 63 (ratio greater than 4 is desirable [22]),suggestive of an adequate signal to noise ratio and therefore the model is significant for the separation process. The % C.V. of all the models were found to less than 10% revealed that all the models were reproducible, (model can be considered reasonably reproducible if % C.V is less than 10%).

From the Table 2, the interaction term with the largest absolute coefficients among the fitted models is AB (+0.41) of tR2 model. The positive interaction between A and B is stastically significant (< 0.0001) for tR2.Changing the factor A from low level to high level strongly affect (decreasing order) the tR2 and Rs2. Changing the factor B strongly affect tR2 but little effect on Rs2. So this study indicated that increasing both the factors A and B will reduce the tR2 and Rs2. A high level of factor A and B will give a shorter run time.

Petrubation plots and response surface plots were offered (Figure 3 & 4) for predicted models in order to given a improved understanding of the investigated method. This type of plots represented the effect of an independent factor on a specific response with all other factors assumed constant at a reference point [15]. A steepest slope or curvature represents the sensitiveness of the response to specific factor. Figure 3(a,b) showed the percentage of methanol in mobile phase (A), the most important effect on tR2 and Rs2 than B. Flow rate (B) have slightly less effect on tR2 and Rs2. But both the factors are vital for the shorter run time. Global optimization: In the present study, the identified criteria for the optimization were; resolution between the critical peaks, capacity factor, and elution time. Derringer’s desirability function was used to optimize the two responses with same target [23]. The Derringer’s desirability function(D), is defined as the geometric mean, weights, or otherwise, of the individual desirability functions. The expression that defines the Derringer’s desirability function is:

(2)

Where n is the number of responses and pn is the weight of the responses.Weight of the response is the relative importance of each of the individual functions di. The relative importance pi is a comparative scale for weighting each of the resulting d i in the overall desirability product and it varies from the least important (pi = 0.1) to the most important (pi = 10). Desirability function (D) can take values from 0 to 1. Weights can range from 0.1 to 10. Weights lower than 1 give less importance to the goal, whereas weights greater than 1 give more importance to the goal. In the present study, pi values were set at 1 for all the three responses. A value of D close to 1, indicates that the combination of the different criteria is matched in a global optimum [15]. The criteria for the optimization of each individual response are shown in Table 3. Criteria I have been proposed for the selecting an optimum experimental condition for analysing routine quality control samples. As can be seen under criteria I, the reponses tR2 and Rs2 were minimized in order to shorten the analysis time. Following the conditions and restrictions as mentioned above, the optimization procedure was carried out. The response surface obtained for the global desirability function is presented in Figure 5. From the Figure 5 it can be concluded that there was a set of coordinates producing high desirability value (D = 0.910) were Methanol concentration of 80% and the flow rate of 1.20 ml/min. The predicted response values corresponding to the later value of D were; tR4 = 4.282 and Rs2 = 10.3005.The prediction efficiency of the model was confirmed by performing the experiment under the optimal condition and the corresponding chromatogram was shown in Figure 6.

In order to investigate the predictability of the proposed model, the agreement between experimental and predicted responses for the predicted optimums I are shown in Table 4. The Percentage of prediction error was calculated by Equation (3) [24]. Predicted Error = Experimental – Predicted / Predicted x 100 (3)

Validation: The developed and optimized method was validated as per ICH guidelines 25. Specificity was performed by comparing the peaks observed in sample solution, blank and placebo (synthetic mixtures). No interference was observed. Hence observed peaks in sample solution was the actual peak of DUT and TAM shown that, the method was specific . System performance was developed by system suitability parameters such as retention time, theoratical plates, asymetric factor and resolution were calculated and percentage RSD was found to be less than 2 % indicating the good performance of the system. Under the experimental condition described above, linear calibration curves for both DUT and TAM were obtained in the concentration range of 15-45μg/ml and 12-36 μg/ml respectively and the correlation coefficients (R2) were found to be 0.9999 for DUT and 0.9998 for TAM, indicating that good correlation existing between concentration and peak area(Figure 7 & 8).

Accuracy was performed at various levels of 50% 75%, 100%, 125% and 150% of lablel claim. The amount of DUT and TAM recovered in all the levels were found to be close to 100 %, indicative of good accuracy of the proposed method (table 5 ). Precision study was performed by injecting the sample solution 3 times at 0hrs, 8hrs,16th hrs and 6 times at day-1, day-2,day-3, by different analysts and in different instruments. The amount of DUT and TAM present in sample solution was found to 98-100 % and 99-102 % mg (table 5). % RSD was found to less than 2%. Robustness of the method was determined by small delibrate changes were made in the method parametres such as wavelength (±2nm), flow rate (±0.2ml) and mobile phase ratio (±5%). But these changes, not affected the method results indicated that the method was robust (table 5 ). Standard and sample Solutions stability were checked up to 3 days at room temperature and the reponses were measured at one time on each day. Results revealed that there was no degradation of DUT and TAM.

All the validation parameters results (Table 5) were indicating that the developed and optimized method was specific, suitable, linear, precise, accurate and robust for the simultaneous estimation of DUT and TAM in pure and pharmaceutical dosage form.

Method apllication to the marketed formulation: Sample solution of the marketed formulation was prepared as per the above procedure as described in the preparation of sample solution. Six replicate injections were given in to HPLC without changing the proposed method procedure. The amount of DUT and TAM present in each tablet was calculated and found to be 0.495mg and 0.402mg respectively (Table 5).

Conclusion:

This developed method is considered as the first method for the simultaneous estimation of DUT and TAM using Chemo metrics assisted RP-HPLC with Photodiode array detection. The various validation characteristics were applied and determined, to assure the suitability of the method.This investigation also proved that, the chromatographic techniques coupled with chemometric tools provide a complete profile of separation process, making this combined technique a powerful analytical tool. Therefore, this validated RP-HPLC-PDA method can bereadily adapted for the simultaneous estimation of DUT and TAM in pure and pharmaceutical dosage form as a routine quality control analysis.

Acknowledgement:

We are thankful to the UGC-BSR, New Delhi, India, for providing financial support to carry out this work.

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