Development and optimization of a simple, robust RP-HPLC technique for analysis of diosmin and hesperidin using quality by design

Quality by design (QbD) is a part of the design of experiments (DOE) that predict the responses using the software. Identification of critical quality attributes (CQAs) is the first step in QbD. The main concept of QbD is the study of dependent parameters as well as the examination of different factors and their interactions. Hence the present study is designed to develop the QbD-based high performance liquid chromatography (HPLC) method and validation of diosmin and hesperidin. The experimental design involves the central composite designs (CCDs) of the reverse phase-high performance liquid chromatography techniques with two factors (mobile phase and pH). The Design Expert software 12.0 version was used to produce optimal chromatographic parameters. Agilent Zorbax SB C 18 column (250 × 4.6 mm, 5.0 μm), the mobile phase used acetonitrile to mono potassium phosphate (formic acid with pH 2.0) (40:60) with a flow rate of 1 ml/minute and retention times 3.434 minutes of diosmin and 5.321 minutes of hesperidin. According to International Conference on Harmonisation criteria, the parameters were validated within the specified limits. The QbD-based HPLC method was developed and validated. The utilization of QbD in the present study leads to more precise and reliable data.


INTRODUCTION
Diosmin is a monomethoxy flavone, a monooxy flavone, a rutinoside, a disaccharide derivative, and a dihydroxy flavanone.It is a bioflavonoid that may be produced from hesperidin or extracted from different plants [1].It is used to treat hemorrhoids and capillary fragility, particularly chronic venous insufficiency (CVI) [2].Hesperidin is a disaccharide derivative, a member of 3′-hydroxy flavanones, a dihydroxy flavanone, a monomethoxy flavanone, a flavanone glycoside, a member of 4′-methoxy flavanones, and a rutinoside.It is functionally related to hesperidin.Hesperidin is a flavanon glycoside found in citrus fruits [3].Hesperidin is most frequently used to treat blood vessel disorders including hemorrhoids, varicose veins, and impaired circulation (venous stasis), either by itself or in combination with other citrus bioflavonoids (such as diosmin) [4].Structures of diosmin and hesperidin are shown in Figure 1 [5,6].
Method development can be an extended process that requires researchers' valuable time.The processes tend to be created utilizing the one factor at a time (OFAT) method, which entails adjusting one variable at a time until the desired result is achieved.This process of method development is systematic, but it takes time [7].A quality by design (QbD) technique uses statistical design of experiments (DOE) to create a "design space" for a robust procedure.The design space defines the experimental region in which changes to technique parameters have no significant effect on the results [8].
analytical quality by design (AQbD) begins with a systematic knowledge of the underlying interaction(s) among the many variables involved in the analysis, followed by early risk assessment studies to identify anticipated essential critical process parameters [9].Following that, factor screening studies are conducted to determine the influential variables, Pipetted 1 ml of the above solutions into a volumetric flask with a 10 ml capacity, then added diluent to the mark (50 ppm of hesperidin, 450 ppm of diosmin).

Sample solution preparation
As per the label claim, the composition of diosmin and hesperidin is, respectively, 450 and 50 mg.52 mg of hesperidin and diosmin sample was precisely weighed and placed into a 10 ml sterilized volumetric flask.The solvent was added, sonicated for up to 30 minutes, centrifuged for 30 minutes to complete dissolution, and then added diluent to the desired volume.After that, the solution was filtered using a 0.45-µ injection filter. 1 ml of the above solution was pipetted into a 10 ml volumetric flask and added diluents to make it the final concentration of 50 ppm of hesperidin and 450 ppm a of diosmin.

Method optimization by applying DOE
The DOE was made using Design-Expert version 12.0 software.(Stat-Ease Inc., Minneapolis, MN, USA).Central composite design (CCD) was used to optimize the method with two variables, and three responses were identified as ideal conditions for the method.13 experimental runs were obtained.

Method operable design region (MODR) establishment
After completing the intended experimental runs in accordance with the CCD, the data were analyzed using regression models and factor-response relationships to produce the MODR.Depending on the provided aim or objective of each critical quality attribute (CQA) on the basis of desirability, the created MODR was used to forecast the optimal chromatographic conditions.

Validation of the optimized method
According to International Conference on Harmonisation (ICH) Q2 (R1) requirements, the RP-HPLC method was validated with different parameters such as system compatibility, linearity, LOD, limit of quantitation (LOQ), intraday precision, inter-day precision, accuracy, and robustness of the presented approach was all thoroughly validated [13,14].

Forced degradation (FD) studies
As per limit of detection (LOD) (Q1A and Q1B) guidelines, FD studies were carried out by exposing the sample to relevant stress conditions like hydrolysis, acid degradation, alkali degradation, oxidation, reduction, thermal, and photolytic degradation were analyzed by HPLC [15].

Preliminary trails
Better separation was observed by using acetonitrile: KH 2 PO 4 in the ratio of 55:45 at 222 nm trails were mentioned in Table 1.

Factors
Based on these preliminary trials, independent and dependent variables were selected.Independent variables are which are then used to optimize the procedure to yield the desired chromatographic solution [10].There are very few analytical techniques are reported for estimating diosmin and hesperidin [11,12].However, no QbD-based reverse phase-high performance liquid chromatography (RP-HPLC) technique for diosmin and hesperidin has been disclosed to date.As a result, the present study was designed for simultaneous QbD-based RP-HPLC estimation of diosmin and hesperidin.The main focus of the goal of the study was to use QbD concepts to create a more scientific and risk-based strategy.for identifying the critical variables for optimizing a stability-indicating HPLC method for diosmin and hesperidin.

Materials
Diosmin and hesperidin were provided by Biocon.All other reagents and chemicals were used HPLC grade procured from Rankem.

Preparation of standard solution
5 mg of hesperidin and 45 mg of diosmin working standards were carefully weighed and placed in a 10 ml sterilized volumetric flask.The solvent has been added and sonicated until completely dissolved.The volume was then brought up to the required level using the same diluent (stock resolution).mobile phase composition and pH, dependent variables are plate count of peak 1, resolution, and tailing factor of peak 1 shown in Table 2.

Experimental runs obtained from CCD
13 experimental runs were obtained from CCD using a 2 3 factorial design.Out of 13 runs, the sixth run was chosen for optimization.Results are shown in Table 3.

Analysis of variance (ANOVA) for quadratic model
The responses were optimized by ANOVA (Table 4).Based on this statistical expression p-value is less than the F-value, which shows an insignificant effect for lack of fit.The p-value should be less than 0.2 it shows a significant effect on the model.Fit statistics represent the adjusted and predicted R 2 values the difference between these two values is less than 0.2.
The residual plots represent the relationship between factors and responses.In R1 (Fig. 2A) the plate count was increased by reducing the mobile phase and increasing the pH. Figure 2B indicated that resolution was increased when decreasing the mobile phase and pH.In R3 (Fig. 2C) the tailing factor was decreased when decreasing the mobile phase and pH.
Based on the Desirability, the optimized chromatographic conditions were selected.The highest desirability showed as 0.869.accuracy, precision, and specificity [16].Summary of validation parameters results is shown in Table 6.

System suitability
According to ICH criteria, all system-relevant parameters have been satisfied and were under the limitations.

Specificity
Any interfering peaks are not observed in blank and placebo chromatograms.Hence, this method was said to be specific.

System precision
Six replicates of standard solutions were injected into the HPLC.
The % relative standard deviation (RSD) of diosmin and hesperidin was found to be 0.18% and 0.33% respectively which indicates the method was precise.

Method precision
The percentage RSD over the areas of six standard injections was within the limits.

Linearity
Linearity was taken in six concentrations starting from 25% to 150% which covers the wide concentration range.The area under the curve for diosmin and hesperidin was determined in the range of 112.5-675 and 12.5-75 µg/ml respectively.The correlation coefficient of diosmin and hesperidin was found to be 0.99975 and 0.99968 respectively.Calibration curves of diosmin and hesperidin are shown in Figure 4.

LOD and LOQ (µg/ml)
LOD for diosmin and hesperidin was found to be 0.405 and 0.045 µg/ml respectively.

Optimized chromatographic conditions
Optimized chromatographic conditions and chromatogram are shown in Table 5 and Figure 3.

Analytical method validation
The optimized method was validated by different parameters like system suitability, linearity, range, LOD, LOQ,  LOQ for diosmin and hesperidin was found to be 1.35 and 0.15 µg/ml respectively.

Table 6 .
Summary of validation parameters.

Table 7 .
FD results for diosmin and hesperidin.