A validated stability-indicating LC method for estimation of etoposide in bulk and optimized self-nano emulsifying formulation: Kinetics and stability effects

Abstract

The present investigation was aimed to establish a validated stability-indicating liquid chromatographic method for the estimation of etoposide (ETP) in bulk drug and self-nano emulsifying formulation. ETP was successfully separated from the degradation products formed under stress conditions on LiChrospher 100 C₁₈ reverse-phase column (a 250 mm × 4.6 mm i.d., 5-μm particle size) using 55:45 (v/v) acetonitrile–phosphate buffer saline (pH 4.5) as the mobile phase, at a flow rate of 1.0 mL min⁻¹ and detection at 283 nm. The response was a linear function of analyte concentration (R² > 0.9997) over the concentration range of 0.05–50 μg mL⁻¹. The method was validated for precision, accuracy, robustness, sensitivity and specificity. The % recovery of ETP at three different levels (50%, 100% and 150%) ranged between 93.84% and 100.06% in optimized self-nano emulsifying formulation, Etosid® soft-gelatin capsule and Fytosid® injection. First-order degradation kinetics of ETP were observed under acidic and alkaline conditions. The method was also applied for the stability assessment of self-nano emulsifying formulation under accelerated conditions, the formulation was found to be stable at all storage conditions with the shelf-life of 2.37 years at 25 °C. The method holds promise for routine quality control of ETP in bulk, pharmaceutical formulations as well as in stability-indicating studies.

1. Introduction

Etoposide (ETP), chemically designated as 4′-demethylepipodophyllotoxin-9-(4,6-O-ethylidene)-β-d-glucopyranoside, is an important antineoplastic agent currently in clinical use for the treatment of small cell lung cancer, testicular cancer and lymphomas (Toffoli et al., 2004; Sissolak et al., 2010). Its mechanism of action involves breakage of DNA strands by reversible interaction with topoisomerase II (Hande, 2008). Low and erratic oral absorption of ETP has been attributed to drug precipitation in the gastrointestinal lumen due to poor aqueous solubility, pH-related degradation and efflux by p-glycoprotein transporter (Patlolla and Vobalaboina, 2005; Tian et al., 2007; Bansal et al., 2009; Akhtar et al., 2011a). In order to overcome the above mentioned constraints, self-nanoemulsifying (SNE) formulation of ETP has been developed and optimized in our laboratory. SNE formulation comprises of isotropic mixtures of natural or synthetic oils, solid or liquid surfactants, or alternatively, one or more hydrophilic solvents and co-solvents/surfactants (Date et al., 2010). A selective and sensitive stability-indicating analytical method is required for the evaluation of ETP based novel drug delivery system. Analytical methods such as high-performance liquid chromatography (LC) coupled with UV detection (Shirazi et al., 2001; Kato et al., 2003; Zhang et al., 2010), fluorescence detection (Robieux et al., 1996), electrochemical detection (Eisenberg and Eickhoff, 1993; Duncan et al., 1986; Cai et al., 1999) and solid-phase extraction (Manouilov et al., 1998) have been previously reported for the determination of ETP. However, most of these methods are not ideal for routine measurements, since they necessitate tedious extraction procedures in case of biological fluids and exhibit long retention times. Modern chromatographic method, such as LC-MS/MS, differential pulse voltammetry and UPLC-qTOF-MS/MS have also been developed to determine ETP level in biological matrixes (Chen and Uckuna, 2000; Radi et al., 2007; Sachin et al., 2010). However, these interfaces are extremely complicated and quite expensive to be employed for routine analysis. Moreover, these are not suitable for parent drug stability test guidelines issued by ICH.

The ICH guideline entitled “Stability testing of new drug substances and products” requires stress testing to be carried out in order to elucidate the inherent stability characteristics of the active substance. These include various stress tests like hydrolytic stability, oxidative stability and photolytic stability testing (ICH, Q1A (R2), 2005). An ideal stability-indicating analytical method should be able to quantify the active constituents and at the same time resolve the drug from its degradation products. This would also enable the detection and measurement of the drug and its degradation products in the presence of excipients employed in the formulation. The literature is silent on the development of a validated stability-indicating assay method in routine analysis of ETP in the presence of its degradation products. Therefore, it was thought necessary to study the stability of ETP under different stress conditions. The main objective of the present manuscript was to develop and validate stability-indicating high-performance liquid chromatographic method for the determination of ETP in the presence of its degradation products in accordance to ICH guidelines. The proposed stability-indicating method is simple and allows rapid for stability studies and quality control analysis of drug in bulk, pharmaceutical formulations as well as in stability-indicating studies. Moreover, the proposed LC method was utilized to investigate the kinetics of this antineoplastic agent under acidic and alkaline conditions at different temperatures and their respective degradation kinetic parameters were calculated with the help of Arrhenius plot. The proposed LC method was also utilized for stability assessment of the optimized self-nano emulsifying formulation under accelerated conditions.

2. Materials and methods

2.1. Materials

Etoposide was (assigned purity: 99.5%) originated as gift sample from the Dabur Research Foundation (Ghaziabad, India). Marketed products (Etosid® soft-gelatin capsule, claimed to contain 50 mg and Fytosid® injection, claimed to contain 20 mg mL⁻¹ of ETP) were commercially purchased. Sefsol 218 was gift sample from Nikko Chemicals (Japan). Transcutol P was kindly provided by Gattefosse, France (Mumbai, India). Cremophor RH 40 was achieved from BASF (Mumbai, India). Triacetin was purchased from E-Merck (Mumbai, India). HPLC-grade methanol and acetonitrile were obtained from Spectrochem Pvt. Ltd. Mumbai, India. Buffers and other chemicals were of analytical-reagent grade. Ultra-pure water was obtained from a Milli-Q system (Millipore, USA).

2.2. Methodology

2.2.1. Standard solution

The stock solution of ETP was prepared by dissolving 10 mg in 20 mL methanol in a 100-mL volumetric flask following dilution to 100 mL with mobile phase. This solution was then diluted as needed to prepare different standard solutions from 0.05 to 50 μg mL⁻¹. The quality control (QC) samples of ETP at three different levels were independently prepared at concentrations of low QC (LQC, 2 μg mL⁻¹), medium QC (MQC, 10 μg mL⁻¹) and high QC (HQC, 40 μg mL⁻¹).

2.2.2. Optimized SNE, capsule and injection preparation

The self-nano emulsifying formulation of ETP was prepared by aqueous titration (low energy emulsification technique) method (Shafiq et al., 2007; Azeem et al., 2009a). The composition of optimized SNE formulation of ETP (ETP_SNE) was 28.57% (w/w) of sefsol 218 and Triacetin (1:1) (oil), 47.62% (w/w) of Cremophor RH 40 (surfactant) and 23.81% (w/w) of Transcutol P (co-surfactant). The optimized ETP_SNE formulation (label claim 50 mg), Etosid® soft-gelatin capsule (label claim 50 mg) and Fytosid® injection (label claim 20 mg mL⁻¹) with an appropriate volume of 300, 200 and 500 μL were suitably diluted with methanol, respectively, to yield a stock concentration of 100 μg mL⁻¹. These solutions were sonicated for 10 min and further diluted with the mobile phase to yield a concentration of 10 μg mL⁻¹. The above solutions were then filtered through a 0.22-μm nylon membrane filter and analyzed in triplicate by proposed LC method.

2.2.3. Instrumentation and chromatographic conditions

The analysis was carried out on a Waters Alliance e 2695 separating module (Waters Co., MA, USA) using photo diode array detector (waters 2998) with autosampler and column oven. The instrument was controlled by the use of Empower software installed with equipment for data collection and acquisition. Compounds were separated at an ambient temperature (25 ± 2 °C), on a 250 mm × 4.6 mm i.d., 5-μm particle size, LiChrospher 100 C₁₈ reverse-phase column with 55:45 (v/v) acetonitrile–phosphate buffer saline (pH 4.5) as the mobile phase at a flow rate of 1.0 mL min⁻¹. Before use, the mobile phase was filtered through a 0.22-μm Nylon filter. The eluent was monitored at a wavelength of 283 nm. The injection volume was 10 μL.

2.2.4. Method validation

Validation was done with respect to various parameters, as required under ICH guideline (ICH, Q2 (R1), 2005). The linearity of the method was confirmed using standard solution of ETP at different concentrations of analytes within the range of 0.05–50 μg mL⁻¹. Each solution was analyzed in triplicate by plotting the peak area against concentration. In method precision, intra-day (repeatability) and inter-day precision and accuracy were determined by six replicate analyses of quality control samples at concentrations of LQC (2 μg mL⁻¹), MQC (10 μg mL⁻¹) and HQC (40 μg mL⁻¹) on the same day and on three consecutive days, respectively. The precision was expressed as the percentage coefficient variation [CV (%)] of measured concentrations for each calibration level, whereas accuracy was expressed as percentage recovery [(ETP found/ETP applied) × 100] (Faiyazuddin et al., 2011). For the determination of % recovery, preanalyzed samples were spiked with 50%, 100% and 150% of the ETP label claim for each amount; six determinations were performed (Akhtar et al., 2011b). This was done to check the recovery of the drug at different levels in the ETP_SNE and in marketed formulations. In addition, the specificity was evaluated by analyzing solutions containing the excipients employed for the preparation of ETP_SNE. The system response was examined for the presence of interference or overlaps with the ETP responses. Robustness was studied to evaluate the effect of small but deliberate variations in the chromatographic conditions at three different levels, i.e. −1, 0, +1. One factor at a time was changed to estimate the effect. The limits of detection (LOD) and quantitation (LOQ) were calculated by the method based on the standard deviation (σ) of the responses for blank samples in triplicate and the slope (S) of the calibration plot, by use of the formulae LOD = 3.3σ/S and LOQ = 10σ/S. The ruggedness of the method was assessed by the comparison of intra-day and inter-day assay results for ETP obtained by two analysts in the same laboratory (Azeem et al., 2009b).

2.2.5. Forced degradation studies

ICH guidelines explicitly require forced decomposition studies to be conducted under a variety of conditions and separation of the pure drug from its degradation products for stability-indicating assay methods (ICH, Q6A, 1999). In forced degradation studies, methanolic stock solution of ETP at concentration of 100 μg mL⁻¹ was prepared. The acid- and base-induced degradations were performed separately by transferring 2 mL of methanolic solution of ETP (100 μg mL⁻¹) to a 10 mL amber colored volumetric flasks to which 8 mL of 0.1 M HCl and 0.1 M NaOH were added, respectively. Both the flasks were sealed and heated under reflux at 80 °C for 30 min. The oxidative degradation was performed by transferring 2 mL of the methanolic solution of ETP (100 μg mL⁻¹) to 10 mL amber colored volumetric flask to which 8 mL of H₂O₂ (20%, v/v) was added. The flask was sealed and heated under reflux at 80 °C for 30 min. UV-degradation was carried out according to option 2 of Q1B in ICH guidelines (ICH, Q1B, 1996). Briefly, 10 mL of methanolic solution of ETP (100 μg mL⁻¹) was transferred in a transparent volumetric flask in a UV light cabinet (Thermo-lab, India) and exposed to radiation at 320–400 nm for 8 h at 25 °C. After removal from the light cabinet, sample was prepared for analysis by diluting to 5 μg mL⁻¹ with the mobile phase. To assess dry-heat-induced degradation, powdered drug was exposed to dry heat at 80 °C/75 ± 5% RH, in a convection oven for 8 h. The bulk drug sample was then prepared for analysis by dilution to 5 μg mL⁻¹ with the mobile phase. All the solutions were filtered through a 0.22-μm nylon membrane filter, before analyzing the solution into the chromatographic system.

2.2.6. Kinetic investigation

The logarithmic values of percentages of the remaining concentrations at different time intervals (0, 5 10, 15, 20 and 30 min) were used to establish the degradation plots of ETP in 0.1 M HCl and 0.1 M NaOH solutions. In brief, 10 mL of methanolic solution (100 μg mL⁻¹) of ETP was mixed with 10 mL of 0.1 M HCl and 0.1 M NaOH, separately, in 100 mL round bottomed (double neck) flasks and heated under reflux at 313, 328, and 343 K for 30 min. The refluxed samples (0.5 mL) were then transferred to 5-mL volumetric flasks and diluted with the mobile phase for analysis. The degradation kinetic parameters such as the degradation rate constant (K_deg), degradation half-life (t_1/2) and shelf-life (t_0.9) at 313, 328, and 343 temperatures (K) were derived from the plots. Each experiment was done in triplicate and data were further processed and degradation kinetic parameters were calculated. The predicted kinetic parameters for the degradation of ETP at 25 °C were extrapolated from Arrhenius plot.

2.2.7. Stability studies

The optimized ETP_SNE (label claim 50 mg) was subjected for stability analysis by charging in stability chamber (Thermo Lab, India) under accelerated conditions (30 ± 2 °C/65 ± 5% RH, 40 ± 2 °C/65 ± 5% RH and 50 ± 2 °C/75 ± 5% RH). Following the protocol, the samples were withdrawn at a time interval of 30, 60 and 90 days and analyzed for % drug content. Analysis was carried out at each time interval by taking 50 μL of each formulation, diluting it to 10 mL with methanol and analyzing by LC system. The amount of drug degraded and the amount remaining at each time interval were calculated. The order of degradation was determined by the graphical method. Degradation rate constant (K_deg) was determined at each temperature. The Arrhenius plot was constructed between log K and 1/T to determine the shelf-life of optimized ETP_SNE formulation, where T is the absolute temperature in degrees Kelvin. The value of K at 25 °C (K₂₅) was obtained by extrapolation of the plot and shelf-life was then calculated by substituting K₂₅ in the following equation:

$$T_{0.9}=0.1052/K_{25}$$

where T_0.9 is the time required for 10% drug degradation is referred to as shelf-life.

3. Results and discussion

3.1. Method development

The chromatographic conditions were optimized with a view to develop a reverse-phase LC stability-indicating assay method. No internal standard was used because no extraction or separation step was involved. The optimization of the method development was done by fixing one variable and changing the other variables among mobile phase composition, flow rate and pH of the mobile phase. ETP is a hydrophobic molecule, almost insoluble in aqueous solvents and freely soluble in organic solvents, such as methanol and acetonitrile. Among the different combinations for solvent system, acetonitrile and phosphate buffer saline of pH 4.5, were preferred as the mixture resulted in a greater response to ETP after several preliminary investigatory runs. The pH of the mobile phase was found to be critical. The peak obtained above pH 4.5, was irregular and asymmetric. Increasing the buffer content enhanced the tailing and decreased the plate count. Changes in concentration of the organic modifier often lead to significant changes in separation selectivity. Increasing the organic modifier content resulted in a decrease in the retention time (R_T) of the drug. Therefore, a high acetonitrile concentration was used at a flow rate of 1.0 mL min⁻¹, keeping in mind the possibility that potential minor degradation products could appear after stress studies and may co-elute with the drug because of the reduced R_T if the flow rate was increased. The peak shape and symmetry were found to be good when a mobile phase composition of 55:45 (v/v) of acetonitrile–phosphate buffer saline of pH 4.5, was used at a flow rate of 1.0 mL min⁻¹ at ambient temperature. Under these conditions, the analyte peak was well-defined with very good symmetry, free from tailing and the R_T of ETP was found to be 4.519 ± 0.017 min. (Fig. 1). The method presents advantages over previously published methods (Manouilov et al., 1998; Eisenberg and Eickhoff, 1993) by the usage of readily available mobile phase acetonitrile and phosphate buffer saline with photo diode array detector and short retention time enabled the analysis of a large number of samples with a small quantity of mobile phase, leading to its cost effectiveness.

Figure 1.

Typical HPLC chromatogram of ETP; R_T at 4.519 ± 0.017 min.

3.2. Method validation

The correlation coefficients (R² > 0.9997) of the calibration plots indicate good linearity in the range of 0.05–50 μg mL⁻¹. The regression equation for the calibration plot was y = 20,903x + 739.47 (n = 3; detection at 283 nm). No significant difference was observed in the slopes of calibration plots prepared on different days (ANOVA, P > 0.05). The intra-day precision was found to be ⩽1.581% (n = 6) and inter-day precision over three different days was calculated as ⩽2.113% (n = 6). Intra-day and inter-day accuracy were found to be 98.00–100.07% and 97.00–100.12%, respectively. The low CV values demonstrated precision of the method (Table 1). The recovery of the method ranged from 93.84% to 100.06% after spiking previously analyzed samples with 50%, 100%, and 150% of additional drug. Low values of RSD ranging from 1.05 to 2.29 also indicate the suitability of this method for the analysis of ETP_SNE formulation and commercially available dosage forms (soft-gelatin capsule and injection) (Table 2). RSD was found to be within limit, which indicated that the proposed method was accurate. There was no interaction observed between drug and excipients present in the nanoemulsifying system, confirmed the specificity of the method. The robustness of the method was investigated under a variety of conditions with respect to changes in flow rate, pH and composition of mobile phase on the analysis of 10 μg mL⁻¹ standard solution samples. The peak area and retention time obtained were in the range of (219,959 ± 1504.919 to 222,802 ± 1926.522) and (4.516 ± 0.023 to 4.518 ± 0.033 min), respectively, following intentional change of flow rate. The effect of change of pH was measured and the peak area and retention time were achieved in the range of (218,335 ± 1682.069 to 228,617 ± 1277.343) and (4.515 ± 0.022 to 4.520 ± 0.024 min), respectively. The results obtained for the peak area and retention time were in the range of (222,561 ± 1707.357 to 222,956 ± 2398.447) and (4.517 ± 0.031 to 4.519 ± 0.027 min), respectively, on deliberate change in the composition of mobile phase. Low RSD values in the range of 0.767–1.099% for these results substantiated the consistency and robustness of the method. Hence, the method was sufficiently robust for normally expected variations in chromatographic conditions. The LOD and LOQ for ETP were determined to be 4.7 and 11.6 ng mL⁻¹, respectively which were quite lower than previously reported method (Shirazi et al., 2001), for which corresponding values were 20 and 40 ng mL⁻¹, respectively. The results strongly advocate that developed method is more sensitive. RSD values for intra-day and inter-day assay of ETP, performed in the same laboratory by two analysts did not exceed 2.0%, indicating the ruggedness of the method.

Table 1.

Precision and accuracy data.

Parameters	Applied (μg/mL)	Found (μg) ± SDa	Precision (CV, %)b	Accuracy (%)c
Intra-day precision	2 (LQC)	1.96 ± 0.031	1.581	98.00
	10 (MQC)	9.99 ± 0.064	0.640	99.90
	40 (HQC)	40.03 ± 0.053	0.132	100.07
Inter-day precision	2 (LQC)	1.94 ± 0.041	2.113	97.00
	10 (MQC)	9.97 ± 0.121	1.213	99.70
	40 (HQC)	40.05 ± 0.087	0.217	100.12

^aMean from six determinations (n = 6).

^bPrecision as a coefficient of variation (CV %) = (Standard deviation/ETP found) × 100.

^cAccuracy = (ETP found/ETP applied) × 100.

Table 2.

% Recovery data.

Formulation (%)	Theoretical (mg)a	Added (mg)	Detected (mg)	Recovery (%)b	RSD (%)
ETPSNE (SNE formulation)
50	50	25	23.46	93.84	2.03
100		50	48.26	96.52	1.62
150		75	75.01	100.01	1.92
Etosid® (soft-gelatin capsule)
50	50	25	23.79	95.16	1.77
100		50	48.21	96.42	1.05
150		75	74.13	98.84	1.92
Fytosid® (injection)
50	20	10	9.46	94.60	2.29
100		20	19.47	97.35	1.53
150		30	30.02	100.06	1.91

^aUnits: mg per formulation.

^bMean from six determinations (n = 6).

3.3. Forced degradation studies

The results from stress-testing studies were indicative of the high specificity of the method. The method was capable of separating degradation products in the presence of pure drug. Well-separated analyte and degradation peaks were obtained in all the degradation experiments (Figs. 2 and 3) such as acid degradation, base degradation, oxidative degradation and photo degradation, respectively. The molecule was found to be labile to acid degradation, base degradation, oxidative degradation and photo degradation with maximum susceptibility to alkaline degradation. In acidic hydrolysis the ETP was found to degrade by approximately 91.53% with one major degradation product (DP1) and one minor product (DP2), which were well separated at R_T 2.057 and 3.527 min, respectively (Fig. 2A). The drug was found to be highly labile to alkaline hydrolysis. On heating the drug solution in 0.1 M NaOH at 80 °C for 30 min, 94.35% degradation was seen with the appearance of two degradation products at R_T 1.823 min and 5.541 min, respectively (Fig. 2B). The drug showed liability to hydrogen peroxide at 80 °C. It decomposed to an extent of 84.97% in 20% H₂O₂ in 30 min. The degradation pattern was found to be similar to alkaline conditions and two degradants were well eluted at approximately 2.191 and 5.532 min, respectively (Fig. 3A). Decomposition was also detected on the exposure of ETP solution to light in a UV-stability chamber with two minor degradation products (DP1 and DP2) at R_T 2.251 and 2.937 min, respectively (Fig. 3B), indicating that the drug sample was also susceptible to UV-photolytic stress. Even under dry-heat conditions, a loss of 17.46% of the ETP was noted within 8 h, which obviously is not a negligible quantity. Summary of the degradation studies of drug is given in Table 3.

Figure 2.

Chromatograms obtained for degradation products of ETP samples. (A) ETP sample degraded in 0.1 M HCl, refluxed for 30 min at 80 °C; degradation product (DP) peaks R_T 2.057 and 3.527 min. (B) ETP sample degraded in 0.1 M NaOH, refluxed for 30 min at 80 °C; degradation product (DP) peaks R_T 1.823 and 5.541 min.

Figure 3.

Chromatograms obtained for degradation products of ETP samples. (A) ETP sample degraded in H₂O₂ (20% v/v), refluxed for 30 min at 80 °C; degradation product (DP) peaks R_T 2.191 and 5.532 min. (B) ETP sample degraded in UV_{320–400 nm}, exposed for 8 h; degradation product (DP) peaks R_T 2.251and 2.937 min.

Table 3.

Forced degradation studies.

Exposure conditions	Applied (μg mL−1)	ETP found (μg)	CV (%)†	Remaining (%)	RT value for degradants⁎	Fig. nos.
ETP-acid [0.1 M HCl; refluxed at 80 °C for 30 min]	10	0.847 ± 0.065	7.67	8.47	2.057, 3.527	Fig. 2A
ETP-base [0.1 M NaOH; refluxed at 80 °C for 30 min]	10	0.565 ± 0.031	5.48	5.65	1.823, 5.541	Fig. 2B
ETP-H2O2 [20% v/v; refluxed at 80 °C for 30 min]	10	1.503 ± 0.172	11.44	15.03	2.191, 5.532	Fig. 3A
ETP-UV [(320–400 nm); exposed for 8 h]	5	3.719 ± 0.481	12.93	74.38	2.251, 2.937	Fig. 3B
ETP-dry heat [exposed at 80 °C/75 ± 5% RH]	5	4.127 ± 0.613	14.85	82.54	2.421	NS⁎⁎

^⁎R_T: value in min.

^⁎⁎NS: not shown.

^†Coefficient of variation (CV %) = (Standard deviation/ETP found) × 100.

The most favorable pH range for ETP stability is pH 4–6. At a pH < 4, hydrolysis of the glycosidic linkage and the lactone ring occurs, and at a pH > 6, epimerization to the less active cis-ETP and intra molecular ester hydrolysis into the salt of the cis hydroxy acid resulted (Holthuis et al., 1989). The degraded products which are produced under stress conditions could be either isomers of parent drug or its hydroxy acid derivatives. It was not the intention of the study to identify degradation products but merely to authenticate that they would not interfere in the analysis if and when present. To conclude, it can be stated that none of the peaks that could be generated by the stress treatment interfered with the peak corresponding to the active component, showing the method is selective and suitable for routine work.

3.4. Kinetic investigation

The kinetic of degradation of ETP was investigated in 0.1 M HCl and 0.1 M NaOH. A constant reduction in the concentration of ETP with increasing time intervals was observed. At the selected temperatures the degradation process of ETP was confirmed by first-order kinetics. The first-order rate constant (K), half-life (t_1/2), and shelf-life (t_0.9) were determined at each temperature for acidic and alkaline degradation (Table 4). First-order kinetics data were fitted to the Arrhenius equation:

$$\text{logK}=\log A-E_{\text{a}}/2.303\mathit{RT}$$

where K is the rate constant, A the frequency factor, E_a the activation energy, R the gas constant (1.987 cal/K/mol), and T the absolute temperature. The Arrhenius plot of log K against reciprocal temperature (1/T × 10³) was found to be linear in the temperature range studied. The degradation rate constants for acidic and alkaline conditions at room temperature (25 ± 1 °C) were calculated to be 0.1854 and 0.2484 h⁻¹, respectively. The first-order t_1/2 and t_0.9 under acidic conditions at 25 °C were found to be 224.28 and 34.04 min, respectively, and for alkaline condition they were found to be 167.58 and 25.44 min, respectively. The first-order degradation kinetics revealed higher values of the degradation rate constants with shorter half-life in 0.1 M NaOH solution compared with 0.1 M HCl solutions. It was concluded that the drug was more susceptible to alkaline degradation, with shorter t_1/2 than for acidic degradation (Table 4).

Table 4.

Kinetic parameters under acidic and alkaline condition.

Condition	Temperature (K)	R	Kdeg (min−1)	t1/2 (min)	t0.9 (min)
0.1 M HCl	313	0.9862	0.0094	73.393	11.141
	328	0.9868	0.0241	28.658	4.350
	343	0.9957	0.0637	10.863	1.649
0.1 M NaOH	313	0.9808	0.0135	51.002	7.742
	328	0.9909	0.0373	18.574	2.819
	343	0.9992	0.1043	6.642	1.018

R: correlation coefficient, K_deg: degradation rate constant.

3.5. Stability studies

Table 5 illustrates slow degradation of ETP at each temperature indicating considerable stability of ETP in the SNE formulation. The order of degradation was determined by graphical method at each temperature which was found to be first order. Therefore, for first order degradation, log% of drug remaining was plotted against time and degradation rate constant (K_deg) was calculated from the slope of the curve at each temperature (Table 5). At 25 °C, the degradation rate constant (K_deg) and shelf-life of optimized ETP_SNE formulation were found to be 1.2173 × 10⁴ (days⁻¹) and 2.37 years, respectively.

Table 5.

Stability study.

Time (Days)	Storage conditions	ETPSNE formulation			Slope × 10−4	Kdeg × 104 (days−1)	t0.9 (year)
		Found (mg)	% Remained	log % remained
0	30 ± 2 °C/65 ± 5% RH	50.00	100.00	2.000	−0.6500	1.4969	1.93
30		49.79	99.58	1.998
60		49.57	99.14	1.996
90		49.33	98.66	1.994
0	40 ± 2 °C/65 ± 5% RH	50.00	100.00	2.000	−1.6612	3.8258	0.75
30		49.42	98.84	1.995
60		48.85	97.70	1.990
90		48.31	96.62	1.985
0	50 ± 2 °C/75 ± 5% RH	50.00	100.00	2.000	−2.0770	4.7834	0.60
30		49.25	98.50	1.993
60		48.62	97.24	1.988
90		47.87	95.74	1.981

RH: relative humidity.

4. Conclusions

A validated stability-indicating LC method was developed for ETP in bulk drug and self-nano emulsifying formulation. All degradants were well resolved from the pure drug with significant differences in their values for retention time. The method was found to be specific as the peaks of the degradation products did not interfere with the ETP peak. Degradation kinetic studies showed higher degradation rates and a shorter half-life under alkaline solution as compared to acidic solution. The optimized nano formulation was found to be stable throughout the analysis at all temperature and humidity conditions. The developed method could be adopted for the routine analysis of samples generated during stability studies of ETP and its formulations.