RP-HPLC Method Development and Validation for Estimation of Naftidrofuryl Oxalate Using Box-Behnken Design

A simple, rapid, precise, sensitive, economical, robust and QbD based RP -HPLC method has been developed for Naftidrofuryl oxalate and validated as per ICH guidelines. The response surface methodology employed with a 3-factor, 3- level Box-Behnken statistical design and used to facilitate method development and optimization. The response surface methodology and multiple response optimizations utilizing a polynomial equation were used to select suitable mobile phase combination. The independent variables studied were the Organic phase (X 1 ), Aqueous phase(X2) and (X 3 ) and the responses was Retention time (Y 1 ), Resolution (Y 2 ) and Tailing factor (Y 3 ). Chromatographic separation was achieved on Zodiac C18 column (100mm×4.6, 3µm),using optimized mobile phase Acetonitrile and pH-7 Tetrabutyl-ammonium buffer solution (90:10), at 282nm. The ﬂow rate and injection volume were 1.0 mL-1 min and 20 µL, respectively. Analysis of variance (ANOVA) confirmed that the three factors were significant. The method was validated for linearity, system suitability, recovery (accuracy), precision, robustness, ruggedness, limit of quantitation (LOD) and detection (LOQ) as per the ICH guidelines. The precision, ruggedness and robustness values were also within the prescribed limits. Calibrations curves were linear (r2= 0.997) at the concentration range of 10 to 50μg/mL. LOD and LOQ values were 12.914µg/mL and 39.132µg/mL respectively. The proposed method was successfully developed by applying QBD based concept and proposed method can be used for routine analysis of Naftidrofuryl oxalate in quality control laboratories.


Introduction
Naftidrofuryl, chemically it is a (RS)-2-(diethylamino) ethyl-3-(1-naphthyl)-2-(tetrahydro furan-2-ylmethyl) propanoate and it is available in the form of oxalic acid. It is also known as nafronyl, INN, NF, NAFTI or as the oxalate salt nafronyl oxalate [1]. The chemical formula of Naftidrofuryl oxalate is (C26H35NO7) with molecular weight is 473.56 as shown in Figure 1 [2]. Naftidrofuryl oxalate shows smooth muscle relaxation, increases cerebral blood flow as well as peripheral blood flow, cerebral adenosine triphosphate concentrations and glucose utilization properties attracts its pharmacological applications in the treatment of senile brain diseases a vasodilator. Therefore, it is widely used in the treatment of peripheral and cerebral vascular disorders [3,4].

Experimental Design
Scouting Step: This step included some trials of the mobile phase that gives an acceptable well resolved chromatographic peak of analyte. At the beginning, different mobile phases containing Tetrabutyl ammonium buffer and Acetonitrile as the aqueous/ organic part of the mobile phase were tried. In addition, change in ratio of Tetrabutyl-ammonium buffer and Acetonitrile were tested.
Finally, the variables that may affect the selected CQA were selected.
Screening Design: In the present study, 3-factor, 3-level Box-Behnken statistical design was used to evaluate the effect of selected responses, to characterize the drug contents and to optimize the developed method. BBD is efficacious for exploration of quadratic response surfaces, mathematical screening and thus helping to optimize the process by using a small number of experimental runs. The dependent and independent variables selected are given Table 1 along with their low, medium and high levels, which were selected based on the results from preliminary experimentation.
The observed responses are given in Table 2.

Ruggedness:
The studies were carried out for two different parameters i.e., Days (Intraday and Interday) and Analyst to Analyst variation. Single concentration level was selected and analyzed as described before three times within the same day (intra-day precision) by different analysts, and on successive three days (inter-day precision). The % RSD was calculated as a measure for method precision.

Scouting Step
An isocratic mobile phase consisting of Tetrabutyl-ammonium buffer 20% and acetonitrile 80% was applied at the beginning. This mobile phase gave strong tailing and no well resolved peak shape.
When % of Tetrabutyl-ammonium buffer was increased and % of acetonitrile reduced, tailing increased strongly. Then we reduced % of tetrabutyl-ammonium buffer and % of acetonitrile increased in order to have better resolution. In this case, good peaks shape was obtained. In this step, three factors were chosen: the % aqueous part of the mobile phase, % of organic phase and flow rate.

ANOVA Technique
The ANOVA (one-way Analysis of Variance) is used to ensure about the significant differences between the means of three or chance that a Lack of Fit F-value this large could occur due to noise.
The average values obtained from the software are given in Table 4.

Main Effects (Lack of Fit)
The Lack of Fit is one of the components of partition of the sum of squares in an ANOVA which can tell that that purpose model is fit or not. The results of Lack of Fit Test and Model Summary Statistics of Retention time (Y 1 ), Resolution (Y 2 ) and Tailing factor (Y 3 ) showed in Table 5.

Interactions
The equation in terms of actual factors can be used to make predictions about the response for given levels of each factors are given in Table 6.

Linearity, LOD, and LOQ
Linearity study was performed on Naftidrofuryl oxalate under the optimized chromatographic conditions; a good linearity was obtained between the peak areas and the concentration ranging from 10 to 50µg/mL. The observations are shown in Table 7, from the data, a plot concentration (µg/mL) Vs Area Under Curve (AUC) was constructed for the Naftidrofuryl oxalate ( Figure 5). The study of graphical plots represents correlation coefficient of Naftidrofuryl oxalate was found to be 0.997.  for system suitability parameters are given in Table 7 and standard chromatogram is given in Figure 6. The correlation coefficient was found to be 0.997 for the drug.

Accuracy
Accuracy of proposed method was as certain on the basis of recovery studies performed by standard addition method. Percent Recovery was performed by preparing the 30 µg/mL solution of Standard Naftidrofuryl Oxalate. The ±S. D and % RSD was calculated and the results for % Recovery is given in Table 8.

Robustness
In these study deliberate changes in the method were done i.e. Change in flow rate (1.0 ± 0.2mL/min), change in mobile phase concentration (organic portion of 90% ± 5%), Change in column (Agilent ZORBAX and ZODIAC) and change in pH of Buffer solution (7.0 ± 0.2 pH) in mobile phase. The robustness results Mean, SD and %RSD of series of measurement were found to be within limit as shown in Table 9.

Ruggedness
The studies were carried out for two different parameters i.e., Days (Intraday and Interday) and Analyst to Analyst variation. In intraday and inter-day variations, results of estimation by proposed methods were found to be varying. In inter-day study% RSD and % estimation was found to be increased after one day storage suggest that the NF is unstable in solution form while in intraday study, solution was found to be stable up to 3 h. The result of estimation for Naftidrofuryl oxalate by different analysts was very much reproducible. This indicates the ruggedness of the method in the hands of different analysts. The results of Ruggedness study for the proposed method is given in Table 10.