INTRODUCTION:

The method for the analysis of drug in the formulation should be robust, sensitive and precise; hence, in this method design of experiment has been applied to study the effect of factors individually and in combination also. Design of experiment (DOE) is based upon the principles of experimental design, mathematical equations or models and outcomes of the factors. This research article focuses on the optimization, development and validation of a new analytical method with DOE^{1, 2}.

Forced degradation/stress testing, defined as the stability testing of drug substance and drug product under conditions exceeding those used for accelerated testing. From a drug development and regulatory perspective, forced degradation studies provide data for the identification of possible degradation products, prediction of degradation pathway, validation of stability-indicating analytical procedures, identification of conditions in which the drug is less stable, the choice of packing material and selection of storage conditions. Although the regulatory guidance documents define the concept of stress testing, they do not provide detailed information about a stress testing strategy. The experimental conditions to conduct stress testing are described in a general way and the exact stress conditions to be applied are not described. Researchers have suggested that degradation can be achieved by exposing the drug, for extended periods of time, to extremes of pH (aqueous, hydrochloric acid or sodium hydroxide solutions) at elevated temperatures, to hydrogen peroxide at room temperature or to UV light and to dry heat (in an oven) while adopting trial and error approach to select the strength, temperature and time of exposure to the stress conditions so as to achieve degradation to an extent of 10–20%, actually. Such trial and error approach are cost, time and labor intensive and should be substituted by more systemic approach^{3, 4}. One such systemic approach is to adopt statistical nested design like factorial design to reveal the variables (strength, temperature or time of exposure) which are most likely to influence degradation and modify only these parameters to effect the adequate degradation.Dutasteride (DUT) is a synthetic 4-azasteroid compound that is a competitive and selective specific inhibitor of both type 1 and type 2 isoforms of steroid 5-α reductase (5AR), an intracellular enzyme that converts testosterone to 5-α dihydrotestosterone (DHT) whereas Tamsulosin (TAM) is α_1a-selective alpha blocker which work by relaxing bladder neck muscles and muscle fibers in the prostate itself and make it easier to urinate^{5- 9}. In this work, an analytical HPLC and stability indicating method has been optimized, developed and validated with DOE for the determination of Tamsulosin and Dutastaride in formulations.

EXPERIMENTAL:

Instrumentation:

HPLC system consisted of a Agilent Technologies, LC-1260 Infinity model connected to PDA (Photodiode array, model no. G1315D 1260 DAD VL with 220 VA was used in this study. Chromatograms were recorded by a computer and treated with the aid of LC Open Lab Software solution. Agilent 5 TC-C18 column (150 mm: 4.6 mm id, 5 µm) was used to perform the separation. The mobile phase was filtered through the Millipore glass filter assembly attached with vacuum pump. The mobile phase was sonicated with Ultrasonic Cleaner. High pure water was produced with a double distillation system, Borosil Corporation.

Chemical and solvents:

The TAM reference standard (RS) was kindly supplied by Gen Pharma (Pune, India) and DUT from Wokhardt Pharma (Aurangabad, India). The formulation, URIMAX D tablet manufactured by Cipla Pvt. Ltd. composed of 50 mg of TAM and 40mg of DUT in each tablet. All chemicals used were of analytical grade and all solvents were of LC grade. Methanol was purchased from SD Fine Chemicals (Mumbai, India). The 0.45 µm pore size Nylon filter papers were purchased from Pall India Pvt. Ltd. (Mumbai, India).

Preparation of mobile phase:

Mobile phase was used in combination of methanol: water ratio 80:20v/v The mobile phase was passed through the filter with 0.22µ pore size and was sonicated for 15 min for degassing before pumping into HPLC system.

Optimization and development of HPLC method:

In the present study, C18 silica packed HPLC column was used for better separation. An Agilent 5 TC-C18 column (150 mm: 4.6 mm id, 5 µm) was used to perform the separation. The solvent proportion, flow rate and wavelength play an important role in HPLC method. Hence, in the present study, these three factors were optimized based on DOE. The mobile phase selected was 75:25, 80:20 and 85:15 ratio of methanol: water. The UV detector was used for detection of drug in the samples. Three different wavelengths such as 231, 233 and 235 nm were studied and one wavelength was selected based on DOE. Similarly, the effect of flow rate (i.e. 0.8, 1.0 and 1.2 mL/min) was studied and based on DOE, the final flow rate was selected. The 20 µl injection volume was used in the study. Most of the formulation studies involve only one variant at a time by keeping others as constant. With the help of full factorial design investigators can study the effect of all the factors by varring them simultaneously.

The factorial design helps to study the effects caused by independent factors and interactions between those self-governing factors. In the present work, three independent factors were used such as solvent composition (A), wavelength (B) and flow rate (C). Three factorial levels were used in the study and were coded as -1, 0 and +1 for low, medium and high, respectively. Totally 27 experimental runs were suggested by the software for analyzing the interaction of each level on formulation characters and the peak area (R1), retention time (R2) and number of theoretical plate (R3) were considered as response factors (dependent factors).

Tables 1 and 2 show the factors chosen and different factor level settings. The significance of independent factors was determined using Fisher’s statistical test for Analysis of the Variance (ANOVA) model that was estimated. The polynomial equation for the experimental design with three factors is given below:

R = βₒ + β₁A + β₂B + β₃C + β₂AB + β₂AC + β₂BC + β₂A²+ β₂B²+ β₂C²

Where R is the response, β is the regression coefficients and A, B and C represent solvent, wavelength and flow rate respectively.

Table 1: Planned 3³ full factorial designs for robustness study.

Independent Factors	Levels
	-1	0	+1
Flow rate (mL/min)	0.8	1.0	1.2
Wavelength (nm)	231	233	235
Solvent	75:25	80:20	85:15

Table 2: 3³ Full factorial designs for robustness study.

Run	Solvent Proportion	Wavelength (nm)	Flow rate (ml/min)
1	75	231	0.8
2	75	231	1
3	75	231	1.2
4	75	233	0.8
5	75	233	1
6	75	233	1.2
7	75	235	0.8
8	75	235	1
9	75	235	1.2
10	80	231	0.8
11	80	231	1
12	80	231	1.2
13	80	233	0.8
14	80	233	1
15	80	233	1.2
16	80	235	0.8
17	80	235	1
18	80	235	1.2
19	85	231	0.8
20	85	231	1
21	85	231	1.2
22	85	233	0.8
23	85	233	1
24	85	233	1.2
25	85	235	0.8
26	85	235	1
27	85	235	1.2

Validation of HPLC method ^{10, 11}:

The developed and optimized method was validated as per the ICH Q2 (R1) guidelines for following parameters such as linearity, accuracy, precision, limit of detection (LOD) and limit of quantitation (LOQ).

System suitability:

For system suitability determination 50 µg/mL and 40 µg/mL of TAM and DUT respectively, was used as stock solution. Six replicate injections of this standard solution were analyzed with HPLC. From these replicate injections, the acceptance criteria for peak area, tailing factor and number of theoretical plate were studied.

Linearity:

The standard calibration curve was prepared with different concentrations from 25-75µg/ml of DUTA and 20–60µg/ml of TAM respectively. Three replicate injections of each concentration were analyzed for this study. The linear regression and correlation coefficient were found out from the graph between peak area and concentration.

Precision:

The precision is a study which is based upon the intra-day precision and inter-day precision. Precision of the developed method was evaluated by performing repeatability (six times of each concentration) of three distinct concentrations two times in a day (i.e. morning and evening) whereas the intermediate precision study was performed by repeating three distinct concentrations on two different days. Intra-day and inter-day precision was performed by using three unusual concentrations. The peak area was measured and percent relative standard deviation (% RSD) was calculated.

Accuracy:

The accuracy of developed method was studied by determining recovery values. The term accuracy means the value which is near to the value of reference. For the determination of accuracy, the standard drug in the range of 80%, 100% and 120% of the sample’s concentration was mixed with sample solution. The same process has been performed in triplicate. The percent recovery of the added standard drug to the assay samples was calculated.

Limit of detection and limit of quantitation:

The sigma method was used for the limit of detection (LOD). This method is based upon the slope and least standard deviation obtained from the response. The formula for the calculation of LOD used was LOD =3.3r/SP, where r is the standard deviation value in response and SP is the slope of the calibration curve. Similarly, the formula for the calculation of LOQ used was LOQ =10r/SP, where r is the standard deviation value in response and SP is the slope of the response.

Analysis of Tablet formulation:

Twenty tablets, labeled as containing 0.5 mg of DUTA, and 0.4 mg of TAM together with excipients were accurately weighed, transferred to a clean and dry mortar and ground into a fine powder. Powder equivalent was accurately weighed, then transferred to a clean 10 ml volumetric flask, 10 ml of mobile phase i. e. Methanol, water (80:20) mixture was added, and the flask was then sonicated for 10 min. Then diluted with mobile phase to give a solution containing 50µg/ml of DUTA and 40 µg/ml of TAM. This solution was filtered through a 0.45µm pore size Nylon 66 membrane filter. The contents of the tablet were calculated using calibration graph or the corresponding regression equation.

Forced degradation study by factorial design ²:

TAM and DUT were subjected to stress under acidic, alkaline, oxidative, thermolytic and photolytic conditions. For acid, alkali, oxidative, dry heat and wet heat conditions, values of variables like time of exposure, temperature and strength were chosen so as to obtain 10–20% degradation. This choice was facilitated by the initial experiments as per the factorial design and performing multiple regression equation to identify conditions for desired 10–20% degradation.

Acid degradation:

1 mg/mL mixture of TAM and DUT in X1 M HCl was heated under reflux at X2°C for X3 min. Two levels were chosen for each of X1, X2 and X3. The high level (+1) for X1, X2 and X3 was 1 M, 75 min and 100°C, respectively, and the low level (-1) for X1, X2 and X3 was 0.1 M, 15 min and 60°C, respectively. Since three variables were considered at two levels, a 2³ factorial design was conducted to set up eight experiments.

Alkali degradation:

1 mg/mL mixture of TAM and DUT in X1 M NaOH was heated under reflux at X2°C for X3 min. Two levels were chosen for each of X1, X2 and X3. The high level (+1) for X1, X2 and X3 was 0.1 M NaOH, 30 min and 100°C, respectively, and the low level (-1) for X1, X2 and X3 was 0.01 M, 10 min and 60°C, respectively. Since three variables were considered at two levels, a 2³ factorial design was conducted to set up eight experiments.

Oxidative degradation:

1 mg/mL mixture of TAM and DUT was maintained in X1%H2O2 in dark for X2 min. Two levels were chosen for X1 and X2. The high level (+1) for X1 and X2 was 30% and 24 h, respectively, and the low level (-1) for X1 and X2 was 3% and 2 h, respectively. Since two variables were considered at two levels, a 2² factorial design was conducted to set up four experiments.

Dry heat degradation:

TAM and DUT powder was spread as a thin film in petri plate and exposed to X1°C for X2 min. Two levels were chosen for X1 and X2. The high level (+1) for X1 and X2 was 200°C and 360 min, respectively, and the low level (-1) for X1 and X2 was 50°C and 30 min, respectively. Since two variables were considered at two levels, a 2² factorial design was conducted to set up four experiments.

Wet heat degradation:

1 mg/mL of TAM and DUT was heated under reflux at X1 °C for X2 min. Two levels were chosen for X1 and X2. The high level (+1) for X1 and X2 was 100°C and 120 min, respectively, and the low level (-1) for X1 and X2 was 60°C and 30 min, respectively. Since two variables were considered at two levels, a 2² factorial design was conducted to set up four experiments.

Photolytic degradation:

TAM and DUT powder was spread as a thin film on petri plate and exposed to direct sunlight for 48 h. A control in dark was also run.

Chromatographic analysis of stressed samples:

Each of the stressed sample obtained was diluted with the mobile phase to get a final concentration of 10 µg/mL, 20µl of the resulting solution was injected on Agilent 5 TC-C18 column (150 mm: 4.6 mm id, 5 µm) at 1 mL/min and the eluent was monitored at 233 nm. The resulting chromatograms were studied for the appearance of secondary peaks and the% reduction in the area of drug peak with reference to standard solution. The% reduction in peak area was considered as %degradation.

RESULT AND DISCUSSION:

Development and optimization of HPLC method:

In this study, the 27 runs were performed, and results were obtained for peak area, retention time and number of theoretical plates. The main interaction plots were created using MINITAB 17 software to estimate the effect of factors on peak area, retention time and number of theoretical plates. Main interaction plots bear the effect of self-governing factors on the dependent factors. Fig. 1 gives the information about the effect of independent factors on retention time. This figure shows that there is no effect of wavelength (B) on retention factor, but solvent proportion (A) and flow rate (C) plays an important role on the same as and when flow rate increases, the retention time decreases.

Fig. 2 gives the information the effect of independent factors on the number of theoretical plate. Figure shows that as the flow rate (C) and solvent proportion (A) increase the number of theoretical plate decreases, whereas with the increase in the wavelength, (B) there is a no effect on the number of theoretical plate.

Fig. 3 is related to the effect of independent factors on peak area. It shows that there is effect of wavelength (B) and solvent proportion (A) on peak area, but the flow rate (C) shows no effect on peak area.

Similarly, the peak area, retention time and number of theoretical plates of each injection were entered in MINITAB 17 software and analyzed using the ANOVA with its significance method. For an experimental design with three variable factors, the suitable model fitting to the data was the quadratic model. The polynomial equations for the response factors are given below:

Rt T = 1.447 A + 0.454 B + 0.126 C - 0.3158 A*A - 0.0608 B*B - 0.0424 C*C - 0.0536 A*B + 0.0064 A*C - 0.0428 B*C

Rt D = 5.412 A + 1.980 B + 0.909 C - 1.084 A*A - 0.295 B*B - 0.169 C*C - 0.189 A*B

- 0.145 A*C - 0.177 B*C

Th Plates T = 8220 A - 4374 B + 1457 C - 2273 A*A + 646 B*B - 858 C*C + 342 A*B + 314 A*C + 517 B*C

Th Plates D = 28217 A - 8105 B + 339 C - 7157 A*A + 1191 B*B - 1249 C*C + 845 A*B + 512 A*C + 648 B*C

Peak Area T = 53344 A + 37819 B + 20363 C - 11371 A*A - 6931 B*B - 3129 C*C - 1606 A*B

- 1666 A*C - 1776 B*C

Peak Area D = 33621 A + 27729 B + 29506 C - 5264 A*A - 4376 B*B - 4376 C*C - 2667 A*B

- 2862 A*C - 2690 B*C

where RtT, RtD, Th Plates T, Th Plates D, Peak area T and Peak Area D are the response factors i.e. retention time, theoretical plates and peak area of tamsulosin and dutastaride respectively. The A, B and C are the solvent, wavelength and flow rate respectively.

Method validation:

Selectivity

The study has been performed as per the ICH Q2 (R1) guidelines (ICH Harmonized Tripartite Guideline and Methodology Q2 (R1), 2005). The chromatogram is shown in Fig. 5. The retention time of the drug was found to be 1.9 min for TAM and 7.9 min for DUT respectively.

System suitability:

System suitability was expressed by percent relative standard deviations of retention time, number of theoretical plate and peak area. System suitability was carried out by performing six replicates. The % RSD value of peak area, tailing factor and number of theoretical plate was found to be less than 0.5%.

Linearity:

The standard calibration curve over the concentration range of 20-60 µg/ml for TAM shows r² = 0.9954 and 25-75µg/ml for DUT shows r² = 0.9919 which is an acceptable value for correlation coefficients. The linear regression from the above gives concentration range that was found to be y= 2719.1x + 2131 and y= 1591.5x + 4842.5 respectively.

Precision:

Precision of new developed method was evaluated by intra-day and inter-day precision and was expressed by percent relative standard deviations of % purity. The %RSD of % purity of intra-day and inter-day precision results is reported in Table 4. The % RSD value of intra-day and inter- day precision was found to be <2%.

Table 4. Intra-day and inter-day precision

Interval		Drug	% purity	SD	% RSD
DAY 1	Morning	TAM	96.07	0.34	0.35
	Morning	DUT	97.18	0.89	0.91
	Evening	TAM	95.42	0.19	0.20
	Evening	DUT	98.36	1.00	1.02
DAY 2	Morning	TAM	96.50	0.39	0.40
	Morning	DUT	97.04	0.66	0.68
	Evening	TAM	96.90	0.95	0.99
	Evening	DUT	97.30	0.91	0.93

Accuracy:

The accuracy study has been performed by the addition of standard drug in samples at three different concentration levels such as 80%, 100% and 120%. The percent recovery at these three different concentration levels was found in the range of 96.7–102.9%. This study suggests a suitable method for the routine experimental analysis of drug in the formulations.

Limit of detection and limit of quantitation:

The limit of detection and limit of quantitation were determined by the sigma method. The limits of detection were found to be 0.15µg/ml and 0.04µg/ml, and limit of quantitation were found to be 0.46µg/ml and 0.13µg/ml for dutastaride and tamsulosin respectively.

Analysis of marketed formulation:

The proposed method was successfully applied to the assay in marketed formulation. The average percentage found was based on the three replicate determinations. The mean % recovery in the examined dosage form was 100.7% and 101.2%. The results obtained were in good agreement with the amount of drug in the labeled claim.

Multiple regression analysis and selection of optimum conditions of force degradation:

The forced degradation experiments set-up on the basis of factorial design were performed and the resulting samples were analyzed by LC. No degradation in drug peak area was observed in case of photolytic, wet heat and dry heat conditions. Substantial degradation was observed in acidic, alkaline and oxidative conditions (Fig. 5). The experimental conditions and degradation obtained for all degradation experiments performed as per the factorial design as summarized in Table 5.

Figure 5 Chromatograms obtained for (A) reference substance, (B) acid hydrolysis, (C) alkali hydrolysis, (D) oxidative degradation (E) wet heat hydrolysis, (F) Dry heat Hydrolysis and (G) photolytic degradation.

Table 5. Experimental design and their resulting % degradation under various conditions.

Expt no	2³factorial design							Expt No	2²factorial design
	X1	X2	X3	Acid Hydrolysis		Base hydrolysis			X1	X2	Oxidative degradation		Dry heat hydrolysis	Wet heat Hydrolysis
				DUT	TAM	DUT	TAM				DUT	TAM
1	-1	-1	-1	15.71	14.33	34.76	24.17	1	-1	-1	20.68	15.68	No Degradation
2	+1	-1	-1	25.42	28.67	43.25	30.12	2	-1	+1	74.69	58.69	No Degradation
3	-1	+1	-1	29.56	28.65	38.25	32.58	3	+1	-1	32.69	29.36
4	+1	+1	-1	35.29	38.35	47.29	42.68	4	+1	+1	82.80	63.23
5	-1	-1	+1	26.38	28.65	33.72	26.28
6	+1	-1	+1	32.68	34.25	36.47	33.85
7	-1	+1	+1	28.5	29.36	38.57	34.58
8	+1	+1	+1	52.41	60.32	65.96	70.17

Further, when results obtained for each experiments performed under acid, alkali, oxidative degradation were subjected to multiple regression, the following equations resulted:

For oxidative degradation, the rough grids of predicted responses were determined from Eq. (3) by considering the values of X1 and X2 from _1 to +1, respectively. From this, it has been observed that 18.75% degradation can be achieved when X1 = 0.75 and X2 = _1. The actual values for X1 and X2 were determined. Thus, when LCZ was kept in 25% H2O2 for 2 h, resulted in 15% degradation. For photolytic conditions about 8% degradation has been obtained.

Acid degradation:

%DEGRADATION OF D = -24.84 + 42.99 CONC OF ACID + 0.7105 TEMP + 0.8393 TIME - 0.7103 CONC OF ACID*TEMP - 0.9259 CONC OF ACID*TIME - 0.01177 TEMP*TIME + 0.01999 CONC OF ACID*TEMP*TIME

% DEGRADATION OF T = -34.33 + 64.13 CONC OF ACID + 0.7945 TEMP + 1.074 TIME - 0.9622 CONC OF ACID*TEMP - 1.435 CONC OF ACID*TIME - 0.01412 TEMP*TIME + 0.02778 CONC OF ACID*TEMP*TIME

Base degradation:

% DEGRADATION OF T IN BASE = 10.87 + 26.72 CONC OF BASE + 0.2678 TEMP + 0.1564 TIME - 0.5478 CONC OF BASE*TEMP - 0.8241 CONC OF BASE*TIME - 0.002302 TEMP*TIME + 0.02210 CONC OF BASE*TEMP*TIME

% DEGRADATION OF D IN BASE = 29.47 + 41.97 CONC OF BASE + 0.1186 TEMP + 0.03048 TIME - 0.6539 CONC OF BASE*TEMP - 1.105 CONC OF BASE*TIME - 0.001097 TEMP*TIME + 0.02231 CONC OF BASE*TEMP*TIME

Oxidative Degradation:

% DEGRADATION OF T = 8.454 + 0.4748 CONC OF H2O2 + 0.1815 TIME - 0.000500 CONC OF H2O2*TIME

% DEGRADATION OF D = 5.363 + 0.5718 CONC OF H2O2 + 0.1466 TIME - 0.001085 CONC OF H2O2*TIME

Analysis indicated that under acidic condition, strength of HCl (X1) and the temperature (X3) were most significant factors and for alkaline condition, time of exposure (X2) and the temperature (X3) were significant. 20% of degradation was calculated using Equations. These equations suggested that for acidic stress, 20% degradation would result by using 0.5M HCl and heating at 40°C for 30 min. When these conditions were adopted in practice, the resulting degradation was 21.36% and 19.45% for tamsulosin and dutastaride respectively. Also, for alkaline degradation would result by using 0.1 M NaOH and heating at 40°C for 30 min. These conditions when adopted in practice 32% and 24 %degradation for tamsulosin and dutastride was achieved. For oxidative degradation, predicted responses were determined from Eq. has been observed that 18.75%and 21.49 % degradation of tamsulosin and dutastaride can be achieved , when kept in 10% H2O2 for 60 min.

CONCLUSION:

The method was successfully developed and optimized through DOE, and data were analyzed using Minitab 17 software. The significant effect of independent factors was analyzed using ANOVA. The design of experiments provides efficient tools for the optimization of variable factors for HPLC method development. Further the method was validated and as per the results, the present method is novel, simple, accurate, precise, economic and robust for the analysis Use of factorial design, expedited the revolution of variables that are most likely to influence the extent of degradation. The use of surface response curves to identify theoretical values of variables for optimum degradation was successful, because when these parameters were put in practice, the % degradation obtained matched the predicted degradation. This suggests that factorial design approach can replace the trial and error approach used to achieve optimum degradation in forced degradation studies.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 16.06.2017 Modified on 12.07.2017

Asian J. Research Chem. 2017; 10(4):504-512.

DOI:10.5958/0974-4150.2017.00082.7

Response	Predicted results	Observed results	Residual values (%)
TAM Rt	1.90	1.99	-4.73
Peak area	114320	124783	-9.15
Number of theoretical plate	7701	8851	-11.03
DUT Rt	7.94	7.94	0
Peak area	84566	85736	-2.96
Numberoftheoreticalplate	24702	26853	-8.70