QUALITY BY DESIGN APPROACH TO DEVELOP STABILITY INDICATING REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT FOR AMBROXOL

Objective: As per requisition of current regulatory requirements, simple, rapid and sensitive method by 33 factorial quality by design approach was established and validated for Ambroxol (AMB) by reversed-phase high-performance liquid chromatography (RP-HPLC).
Methods: A simple RP-HPLC method has been developed and validated with different parameters such as linearity, precision, repeatability, limit of detection (LOD), limit of quantitation (LOQ), accuracy as per International Conference for Harmonisation guidelines (Q2R1). Statistical data analysis was done for data obtained from different aliquots Runs on Agilent Tech. Gradient System with Auto injector, ultraviolet (UV) diode-array detection and Gradient Detector.
Results: Equipped with Reverse Phase (Agilent) C18 column (4.6 mm × 100 mm; 2.5 μm), a 20 μl injection loop and UV730D Absorbance detector at 244 nm wave length and running chemstation 10.1 software and drugs along with degradants were separated via Methanol: (0.1% orthophosphoric acid) Water (75:25) of pH 3 as mobile phase setting flow rate 0.7 ml/min at ambient temperature the retention time of AMB were found to be 4.85 min. The industrialized method was found linear over the concentration range of 10–50 μg/ml for AMB while the LOD and LOQ of AMB was found to be 0.5174–0.2739 μg/ml, analytical method that concluded.
Conclusion: There are no interfering peaks underperformed degradation conditions. Therefore, a sensitive, robust, accurate, and stability indicating method was developed with high degree of practical utility.


INTRODUCTION
The concept of "quality by design" (QbD) was defined as an approach that covers a better scientific understanding of critical process and product qualities, designing controls and tests based on the scientific limits of understanding throughout the development phase and by the knowledge obtained during the life-cycle of the product to work on a continuous improvement environment. QbD describes a pharmaceutical development approach referring to formulation design and development and manufacturing processes to maintain the prescribed product quality. Guidelines and mathematical models are used to ensure the establishment and use of the knowledge on the subject in an independent and integrated way [1][2][3].
Ambroxol hydrochloride (HCL) [4,5] is a potent mucolytic and mucokinetic. Ambroxol (AMB) HCL is chemically Trans-4-([2-amino-3, 5-dibromobenzyl] amino)-cyclohexanol HCL (Fig. 1). AMB is indicated as "secretolytic therapy in bronchopulmonary diseases associated with abnormal mucus secretion and impaired mucus transport. It promotes mucus clearance, facilitates expectoration, and eases productive cough, allowing patients to breathe freely and deeply." Moreover, the extensive literature inspection revealed that there is no reversed-phase high-performance liquid chromatography (RP-HPLC) method obtainable for simultaneous estimation of AMB in the pharmaceutical dosage forms using experimental design approach QbD. A few analytical methods [6,7] have been reported in the literature for the determination of levofloxacin or AMB alone or combination with other drugs biological fluids, and pharmaceutical dosage forms. They include derivative spectrophotometric [8,9] methods and other methods such as HPLC with ultraviolet (UV) detection, high-performance thinlayer chromatography [10,11], and liquid chromatography with tandem mass spectrometry were also reported for the determination of AMB from human body fluids and pharmaceutical dosage form.

Regulatory authorities such as Food and Drug Administration and
International Conference for Harmonisation (ICH) guidelines [12][13][14] are promoting and requesting the application of experimental design approach to understand chromatographic selectivity and support better method control, including method transfer. This prompted the investigators to adopt the experimental design in HPLC, and many papers were published related to this work [15][16][17][18]. The main impartial of our work is to develop an improved RP-HPLC method suitable for the routine quality control of AMB in a pharmaceutical industry and provide information on the sensitivity of chromatographic factors and their interaction effects on the separation characteristics. In our studies, no method has been reported for simultaneous estimation of the AMB drugs by using QbD based 3 3 factorial designing.

Chemicals and reagents
Reference standards of Naproxen HCL were obtained as gift sample from R.S.I.T.C Jalgaon, India. Pharmaceutical formulation was purchased after local market (Brand: Ambrolite tablet labeled claim AMB 30 mg tablets India Ltd. 100 tab). The HPLC grade solvents used were of E-Merck (India) Ltd., Mumbai. HPLC grade Methanol and Ortho Phosphoric Acid (Merck, Mumbai, India) were used in the analysis. HPLC grade water was prepared using Millipore purification system.

Instruments
The analysis of the drug was conceded out on Agilent Tech. Gradient System with Autoinjector, UV diode-array detection and Gradient Detector. Equipped with Reverse Phase (Agilent) C 18 column (4.6 mm × 100 mm; 2.5 µm), a 20 µl injection loop and UV730D Absorbance detector and running chemstation 10.1 software.

Preparation of std. AMB solution: (Stock I)
An accurately weighed quantity, 10 mg of AMB was dissolved in methanol in a 10 ml volumetric flask and volume made up to 10.0 ml to produce a solution of 1000 µg/ml. From the freshly prepared standard stock solution (1000 µg/ml), 0.1ml stock solution was pipetted out in 10 ml of volumetric flask, and volume was made up to 10 ml with mobile phase to get final concentration of 10 µg/ml.

Preparation of std. AMB solution: (Stock III)
From the freshly prepared standard stock solution (1000 µg/ml), 0.1 ml stock solution was pipette out in 10 ml of volumetric flask, and volume was made up to 10 ml with mobile phase to get final concentration 10 µg/ml.

QbD approach to analysis
The application of QbD in HPLC method growth commences with establishing analytical objectives based on sound science to ensure consistent method presentation characteristics are achieved. The use of QbD for an analytical way begins with defining the target analytical profile in which the pre-defined objectives for method performance must be appropriately validated and documented.
Thus the objective of this work was to perform experimental design by using Design Expert Software leading to develop simple, rapid, and sensitive method by QbD approach and validated as per ICH Guidelines (Q2R1) for Naproxen and Pantoprazole and its stability indicating method by RP-HPLC. Further statistical data analysis to be done along with numerical and graphical optimization to develop Analytical Design Space.

Calibration curve
A calibration curve was constructed succeeding replicate (n=6) analysis of five standards of 10, 20, 30, 40, and 50 µg/ml of AMB. The peak height ratio of drugs was calculated and plotted area under the curve (AUC) versus concentration after which least squares linear regression analysis of data was undertaken to establish the equation for the best fit line and the correlation coefficient (R 2 ) to authorize linearity. Samples were injected and peaks were recorded at 244 nm and the graph plotted as concentration of drug versus peak area as shown in (Table 1 and Fig. 3).

Precision
Intra-day (repeatability) precision was established following analysis of replicate samples (n=6) at three concentrations indicative of low, medium, and tall levels within the linear range viz., 20, 30 and 40 µg/ ml of AMB. Analysis was performed over a small period of time on the same day. Inter-day precision or reproducibility was assessed at low, medium, and high concentration on three consecutive days and the percent relative standard deviation (% RSD) was used to assess intra-and inter-day precision. An upper limit of 2% was used to confirm precision in our laboratory. Precision of an analytical method is usually expressed as standard deviation or RSD (Table 2).

Accuracy
Recovery studies were performed to validate the accuracy of developed method. To pre-analyzed tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed. Statistical validation of recovery studies shown in (Table 3).

Specificity
The specificity of an analytical method is defined as the ability of a method to ensure that the peak(s) of interest elute as distinct responses in the presence of excipients, impurities or degradation compounds.

Robustness
To evaluate robustness few parameters were deliberately varied. The parameters include variation of flow rate, percentage of methanol as described in (Table 4).

Study of system suitability parameters
The system suitability is used to verify, whether the resolution and reproducibility of the chromatographic system are adequate for analysis to be done. The test was performed by collecting data from five replicate injections of standard solution as shown in (Table 5).   (Fig. 4) the amounts of AMB per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with tablet formulation. Tablet Assay for %Label claim for % RSD Calculated, Result was shown in (Table 6).

Forced degradation studies
Forced degradation study was performed to evaluate the stability of the developed method using the stress conditions such as exposure of sample solution to acid, base, Hydrogen peroxides (H 2 O 2 ), and Neutral. Investigation was done for the degradation products in different conditions and are shown in (Table 7).

Acid hydrolysis
The acid hydrolysis performed using 0.1N HCl at 70°C for 1 st h for AMB to indicate degradation. The major degradation product for AMB was observed at relative RT (RRT) for 1 st h.

Alkaline hydrolysis
The alkaline hydrolysis condition was performed using 0.1N NaOH at 70°C for 1 st h for AMB. The major degradation product for AMB was observed at RRT for 1 st h.

Oxidation
In the oxidation condition with 3% H 2 O 2 for 1 st h AMB show oxidative stress degradation peak in the chromatogram.

Neutral
There was no major degradation observed for AMB and hence they were not sensitive to light at 70°C for 1 st h.

RESULTS AND DISCUSSION
Such analytical methods are, in fact, an indicator of a quality product and the robustness of that product for the duration on the lifecycle of that product. The main goal of any HPLC method is to separate and quantitate analyte(s) of interest from any impurity and/or excipients. Initially, it is important to establish the critical quality attributes of a system that may impact the quality of the analytical method. Development of Analytical RP-HPLC Method with Design Space and Control Strategy determination by optimization study all the computations for the current optimization study and statistical analysis were performed using Design Expert® software (Design Expert trial version). State-Ease Inc., Minneapolis, MN, USA).

Application of design of experiments (DOE-1) for method optimization DOE-1
Thus, 3 randomized response surface designs with a full fraction design were used with 17 trial runs to study the impact of three factors on the three key response variables. In this design three factors were evaluated, each at three levels and experimental trials were performed at all three possible combinations. The mobile phase composition (χ 1 ), Wavelength (χ 2 ), and flow rate (χ 3 ) were selected as independent variables and RT and Resolution were selected as dependent variables. The resulting data were fitted into Design Expert 10 Software and analyzed statistically using analysis of variance (ANOVA) and F-Test (Fig. 5) indicates the normal plot of residuals for RT with other chromatographic parameters. The data were also subjected to 3-D response surface methodology to determine the influence of flow rate, Wavelength, and mobile phase composition on dependent variables as shown in (Fig. 6). The probable trial runs using 3 3 full fraction designs are as shown in (Table 4). Further ANOVA and F-test with variables are shown in (Tables 8-11). Moreover degradation peaks of API were shown in (Figs. 7-10) from acidic, alkaline, peroxide, and Heat.

CONCLUSION
A simple, rapid, reliable, robust, and optimized reversed phase highperformance liquid chromatographic method for estimation of Naproxen and Pantoprazole was successfully developed and validated as per ICH guidelines. Percentage of mobile phase, flow rate, and wavelength was optimized by using QbD approach i.e. 3 3 factorial design. There are no interfering peaks underperformed degradation conditions. Therefore, a sensitive, accurate, and stability-indicating method was developed with high degree of practical utility.