Development and validation of simple RP-HPLC-PDA analytical protocol for zileuton assisted with Design of Experiments for robustness determination

2.1 Drugs and reagents

Zileuton standard was obtained as a gift sample from Biophore India Ltd. (Hyderabad, India), (85% w/v) orthophosphoric Acid (OPA), Hydrogen peroxide (30% w/v), Sodium starch glycolate (SSG) and Micro-crystalline Cellulose (MCC) were procured from Loba Chemie, India. Methanol (HPLC Grade), sodium hydroxide (NaOH) and hydrochloric Acid (HCl), were purchased from Merck Ltd., India. Double distilled water was used throughout the analysis.

2.2 Equipments and experimental conditions

Analysis was performed on UFLC – LC 20 AD (Shimadzu Corporation, Japan) consisting of LC – 20 AD binary solvent delivery system (pump), SPD-M20A diode array detector and CTO – 10 AS vp; column oven, a rheodyne injector with 20 μl loop and a Hamilton syringe (100 μl). Separations were achieved on a LC – GC Qualisil BDS C18 column (250 mm × 4.6 mm, 5 μm). Data collection and analysis were performed with LC-solution (Shimadzu Corporation, Japan). Stress degradation studies were assisted with i-Therm® AI-7981, thermostatic Water Bath with digital controller. All weighing operations for the present analysis were carried out with the help of SHIMADZU AUX-120 analytical balance. Ultrasonication of samples was performed using Ultrasonicator; ENERTECH Electronics Pvt. Ltd., India.

2.3 Preparation of in-house zileuton tablets

As the tablet formulation was not available in Indian market; tablets containing 600 mg of zileuton were prepared in-house using direct compression technique and employing SSG as super disintegrant and MCC as diluent. Prepared tablets were used as pharmaceutical formulation for further analysis.

2.4 Preparation of stock standard solution and study of calibration curve

Stock solution of zileuton was prepared with a concentration of 100 μg mL⁻¹ in a mixture of methanol and (0.2% v/v) OPA (80:20 v/v). Determination of linearity involved analysis of six working solutions having concentrations 2 μg mL⁻¹, 4 μg mL⁻¹, 6 μg mL⁻¹, 8 μg mL⁻¹, 10 μg mL⁻¹ and 12 μg mL⁻¹, respectively; obtained by serial dilution of stock standard solution with mobile-phase. Resulted peak areas and concentrations were subjected to regression analysis to establish a relationship as calibration curve.

2.5 Preparation of sample solution

The sample solution was prepared from in-house formulated zileuton tablets. The quantity of pulverized tablet mass equivalent to 50 mg zileuton was transferred into 100 mL volumetric flask containing 25 mL of methanol, after ultrasonication for 20 min; volume was made up to the mark to get the concentration of 500 μg mL⁻¹. The resulting solution was filtered through a 0.45 μm filter (Millifilter, Milford, MA, USA). From filtrate, appropriate volumes of solution were transferred using micropipettes into 10 mL volumetric flasks and the volume was made up to the mark with mobile phase to obtain the final concentrations of 8 μg mL⁻¹. Resulting solutions were subjected to proposed method for further analysis.

2.6 Chromatographic conditions

2.7 Stress degradation studies for zileuton

The optimized LC method was used to study the degradation behaviour of the drug under various stress conditions. Stress studies were carried out as per ICH Q1A (R2) recommendations for hydrolysis, oxidation, thermal (dry heat stress) and Q1B recommendations for photolysis. The stressors, choice of its concentration and preparation of samples were based on pre-developed laboratory protocol. The optimized stressed conditions are enlisted in Table 1.

As the drug was insoluble in water; hydrolytic stress was induced by dissolving 10 mg of drug in methanolic solution of stressor in a 10 mL volumetric flask and volume was made up to the mark. Resulting solution was transferred into a 50 mL round bottom flask (RBF); fitted with a reflux condenser and refluxed for specified period. Sample solution (0.1 mL) was withdrawn with a 1.0 mL pipette and neutralized. Volume of resulting solution was made up to 10 mL using mobile phase; in a 10 mL volumetric flask. Solution thus obtained was then subjected to chromatographic determination. Sample solutions from all stressed situations were diluted with mobile phase to achieve a final concentration of 10 μg mL⁻¹ before injecting into HPLC system.

3.1 Accuracy

Accuracy of the method was evaluated by spiking the drug standard in predetermined tablet solution at concentration levels of 80%, 100% and 120% and determined as percent recovery studies.

3.2 Precision

The precision for an analytical method illustrates information on the random errors. It represents the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample under optimized conditions. It is divided into repeatability (intra-day precision), intermediate precision (inter-day precision).

Intra-day and inter-day precisions for present RP-HPLC-PDA analysis were determined by analysing, three different concentrations 4 μg mL⁻¹, 6 μg mL⁻¹ and 8 μg mL⁻¹ using three repetitive measurements at each target concentration level.

3.3 Limit of detection (LOD) and limit of quantitation (LOQ)

The determination of LOD and LOQ was based on the average standard deviations of the responses and slopes of constructed calibration curves (n = 3) as described by ICH guidelines Q2 (R1). Hence sensitivity of the proposed method was estimated in terms of LOD and LOQ using formulae; LOD = 3.3 × ASD/S and LOQ = 10 × ASD/S; where, ‘ASD’ is the average standard deviation and ‘S’ is the slope of corresponding calibration curve. LOD and LOQ were estimated at lower range of calibration curve in between 2 μg mL⁻¹ and 4 μg mL⁻¹ at concentrations of 2 μg mL⁻¹, 2.5 μg mL⁻¹, 3.0 μg mL⁻¹, 3.5 μg mL⁻¹, and 4.0 μg mL⁻¹.

3.4 Robustness and experimental design methodology for robustness

Method robustness was evaluated as per ICH to determine the constancy of the results when variables inherent to the method of analysis are varied deliberately and it depicts the reliability of the analytical procedure opted. Robustness testing was performed in order to obtain information about those critical parameters affecting the response (peak area, retention time and found concentration). To study the simultaneous variations of the factors on the considered responses, a multivariate approach DOE with CCD was applied. Simultaneous variations of the factors can be studied effectively using DOE approach which was applied efficiently to test method robustness. CCD was employed to obtain predictive model describing the changes in the responses within the experimental domain.

A CCD in ‘k’ factors require ‘2^k’ factorial runs, ‘2k’ axial experiments, symmetrically spaced at ±α along each variable axis and at least one centre point. Two or five Central repetitions are generally carried out in order to know the experimental error variance and to test the predictive validity of the model (Lunsted et al., 1998). Statistical validation of polynomial equations generated by Design Expert® software (version 8.0.1, Stat-Ease Inc., Minneapolis, MN) was established by analysis of variance (ANOVA) and coefficients of second order quadratic model were estimated by least square regression. Multiple linear regression statistical models result in predictor equations incorporating interactive and polynomial terms for evaluation of mutual relationships between the input variations and output responses; quoted as second order polynomial with the form:

Three dimensional surface responses were plotted to easily and more precisely define the effect of variations on method robustness which allowed predicting the behaviour of analyte slightly outside the experimental domain.

3.5 System suitability study

According to the United States Pharmacopeia, system suitability tests are integral part of liquid chromatographic methods. Retention time, capacity factor, number of theoretical plates, and asymmetry factor were calculated for standard solutions. The values for retention time (t_R), capacity factor (k′), theoretical plates (N) and asymmetry factor obtained from system suitability study were found to be 4.201 min, 1.356, 5431.475 and 0.334, respectively. The data was found to be within acceptable limit.

4.1 Optimization of chromatographic conditions

Different mobile phases were tested with a view to achieve simple, rapid and economical separation between drug and degradation products. The optimal mobile phase composition was found to be methanol (0.2% v/v) and orthophosphoric acid (80:20 v/v) and the run time was 10 min. Before analysis, both the mobile phase and sample solutions were filtered through a 0.45 μm membrane filter and degassed for 15 min in ultrasonicator. Chromatographic studies were performed at ambient temperature. The optimized flow rate was 1.0 mL min⁻¹ with an injection volume 20 μl followed by detection at wavelength of 260 nm.

4.2 Linearity study

Determination of linearity and establishment of calibration curve involved plotting graph between peak areas obtained versus concentrations. Linear relationship was obtained for the concentration range of 2–12 μg mL⁻¹ with a slope ± SD of 50,654 ± 183.22, intercept ± SD of 2296 ± 45.88 and correlation coefficient ± SD of 0.999 ± 0.0001. The regression equation obtained during determination of linearity was, y = 50,654 x + 2296.

4.3 Method validation

4.3.4

4.3.4 Robustness and design analysis for robustness

Three factor face centred design (FCD) consisting of eighteen experiments including four replicates at each centre point was used. All experiments were performed in randomized order to minimize the effects of uncontrolled factors that may introduce biased responses. Rather than analysis of single coefficient whole model equation was used and for response surface analysis; crucial focus was given to factors whose responses are with or without significance and are considered too. Equations obtained from the model were with the forms:

(2)

$$y_1=1216972-5743.39x_1-768191x_2+78179.93x_3-3085.25x_1{x}_2-2750.25x_1{x}_3+71087.5x_2{x}_3+57.06x_1^2+308221.40x_2^2+169071.40x_3^2$$

(3)

$$y_2=49.39-0.67804x_1-24.1705x_2-6.2119x_3+0.2405x_1{x}_2+0.1765x_1{x}_3-8.5x_2{x}_3+0.002072x_1^2+0.879762x_2^2-0.47024x_3^2$$

(4)

$$y_3=299.6321-1.41265x_1-189.705x_2+19.25577x_3-0.76072x_1{x}_2-0.67807x_1{x}_3+17.51125x_2{x}_3+0.01405x_1^2+76.10569x_2^2+41.76419x_3^2$$

Estimation of experimental error and measurement of validity of polynomial models (lack of fit) were obtained through repetition of experimental points (optimized level of variables). ANOVA was used to obtain regression lack of fit. As the calculated F-ratios were not greater than the tabled F-values at 95% confidence level there was no evidence of models lack of fit and the models were provided with adequate representation of data. Taking into account the degrees of freedom it was indicated that the data is well fitted to regression models as depicted in Table 4 (Ferreira et al., 2007).

Table 4 The estimates of CCD regression analysis and statistical parameters of ANOVA for robustness studies of zileuton.

Statistical parameters	y1: Area	y2: Retention time	y3: Conc. of OPA
R2 a	0.9925	0.9782	0.9925
R2 adj.	0.9841	0.9537	0.9841
SDb	3752.5	0.1046	0.9258
C.V.%c	3752.5	2.4594	0.9157
DFd	9	9	9
SSe	14949836154.738	3.9335274801587	910.43549734121
MSf	1661092906.0821	0.43705860890653	101.15949970458
F-ratiog	117.964849	39.94223812197	118.02000249801
p-value	<0.0001	<0.0001	<0.0001

a Coefficient of regression.

b Standard deviation.

c Coefficient of variations.

d Degrees of freedom.

e Sum of squares.

f Mean sum of squares.

g Fischer’s ratio.

As per the values of coefficients from the polynomial models and their signs (Eqs. (2)–(4)), x₁ (methanol content) and x₂ (flow rate) have negative influence on the responses; y₁ (peak area), y₂ (retention time) and y₃ (found concentration) while x₃ (concentration of OPA) has the positive effect on y₁ and y₃ and negative effect on y₂. Response surfaces from DOE revealed linear models (suggested) for all the three variables (x₁, x₂ and x₃) and depicted least influences of the methanol content and concentration of OPA while flow rate showed the highest influence on area, retention time and found concentration; respectively. When concentration of OPA was kept constant; the increase in the content of methanol in mobile phase and flow rate leads to decrease in the peak area and retention time and increased found concentration of the analyte. Steepness of the response surface plots (Fig. 2A–I) demonstrates that when one of the factors was held constant at a specified level, usually the proposed optimum; deliberate change in the flow rate; affected peak area (y₁) more as compared to other two factors (Fig. 2A and B). On the other side keeping flow rate constant (Fig. 2C, F, I) resulted in no considerable change in peak area and found concentration while retention time deviated slightly even though the methanol content and concentration of OPA varied notably; decrease in flow rate increased area and vice versa. Increase in the flow rate resulted in decreased found concentration and retention time (Fig. 2D, E, G, H), while the methanol content and OPA had no prevailing effect over the flow rate of mobile phase.

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Figure 2

3D response surface plots for Area – peak area (y₁), RT – retention time (y₂) and FC – found concentration (y₃); against flow rate vs. methanol content – (A), (D), (G); flow rate versus concentration of OPA (B), (E), (H) and concentration of OPA versus methanol content – (C), (F), (I).

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4.4 Stress degradation studies

Degradation products with their respective retention time (t_R) and optimized stress conditions are represented in Table 1. Chromatogram for zileuton standard (Fig. 3A) depicted a single major peak at 4.0 min. Acidic hydrolysis resulted in three degradation products represented as III, VII and IX (Fig. 3B), four degradation products II, III, IV, and VII; were produced during basic hydrolysis (Fig. 3C), neutral hydrolysis showed four degradation products II, III, IV, and VII (Fig. 3D). Oxidative stress in 3% H₂O₂ (Fig. 3E), formed three degradation products I, IV, and VI while photolysis on exposure of drug in acidic medium produced degradants IV, VI and XII (Fig. 3F), in alkaline medium V, VII, VIII and XI (Fig. 3G), in neutral medium (Fig. 3H) only VI and XIII and in solid state stress (Fig. 3I) resulted in degradants I, III and VI. Exposure to dry heat (Fig. 3J) produced four degradants I, III, VII and X.

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Figure 3

(A–J) Chromatograms, showing zileuton and its degradation products in various stress conditions applied (I–XIII are zileuton degradants in different stress conditions).

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