Kinetic study of the alkaline degradation of imidapril hydrochloride using a validated stability indicating HPLC method

Abstract

An aqueous alkaline degradation study was performed for imidapril hydrochloride (IMD) drug in the presence of its degradation products and an isocratic stability indicating method was presented using a HPLC technique. The separations were performed using an ACE Generix 5C8, 150 × 4.6 mm column and a mobile phase consisting of buffer solution (0.1 M potassium dihydrogen phosphate and 0.02 M tetra-N-butyl ammonium hydrogen sulphate of pH = 4.5 with 1 N HCl) and acetonitrile 60 : 40 (v/v). The wavelength of the detector was adjusted at 210 nm. The method showed high sensitivity concerning accuracy, precision, linearity and specificity within the acceptable range from 0.1 to 100 μg mL⁻¹ and the limit of quantification was found to be 0.0211 μg mL⁻¹ for IMD. The proposed method was used to determine the drug in its pharmaceutical formulation and to investigate the degradation kinetics of the drug's alkaline-stressed sample. The reactions were found to follow a first-order reaction. The activation energy could also be estimated. The optimized stability indicating HPLC method was validated according to ICH guidelines.

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1. Introduction

Imidapril hydrochloride (IMD) has the IUPAC name of (4S)-3-[[(2S)-2-[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl] amino]-1-oxypropyl]-1-methyl-2-oxo-4-imidazolidine-carboxylic acid monohydrochloride. Its chemical formula is C₂₀H₂₇N₃O₆·HCl and the molecular weight is 441.91 g mol⁻¹ for the IMD salt.¹ It is a recently developed prodrug-type angiotensin-converting enzyme (ACE) inhibitor, (Fig. 1).

Fig. 1 Chemical structure of imidapril hydrochloride.

Clinically, IMD is used in the treatment of hypertension, chronic congestive heart failure and acute myocardial infarction. Unlike other ACE inhibitors, IMD has the advantage of being associated with lower incidence of dry cough. It is rapidly absorbed and metabolized in the liver to imidaprilat which is twice more potent than captopril (a well-known ACE inhibitor). Imidaprilat plasma level increases gradually and slowly declines.²

A literature survey regarding imidapril analysis revealed that there are several methods, which are based on different techniques such as UV spectrophotometry,^3–5 HPLC and GC,^6–15 an enantioselective chiral LC method¹⁶ and TLC-densitometric method.¹⁷ In modern analytical laboratory, there is always a need for significant stability-indicating methods of analysis. The focus of the present study was to develop and validate simple and accurate stability indicating method for the quantification of IMD in bulk form or in the presence of IMD alkaline-degradation. Moreover, kinetic studies and accelerated stability experiments are important to solve problems encountered in quality control and to predict the expiry dates of pharmaceutical products. So, our aim in this work was to develop simple, accurate, specific and reproducible stability-indicating method for the determination of IMD in the presence of its possible degradation products. The method aimed to include kinetic studies, which are important for the quality control of pharmaceutical products. The proposed methods were developed, validated¹⁸ and compared to a reported HPLC method.

2. Materials and methods

2.1. Instruments

A liquid chromatograph consisting of an Agilent HPLC instrument (Agilent, USA) 1200 series Rapid Resolution (RR), Binary pump SL (Model G1312B Agilent 1200 series; Agilent Technologies) connected with auto sampler SL (Model G1329B Agilent 1200 series; Agilent Technologies), Thermostat column compartment SL (Model 1316B, Agilent 1200 series; Agilent Technologies) equipped with a diode array detector DAD SL (Model G1315C, Agilent 1200 series; Agilent Technologies), data acquisition and analysis was performed on Chemstation module. Metler Toledo balance (AB2665S Model, Switzerland), digital pH meter (HANNA PH211 Model, Romania), vacuum filter pump (Model XI 5522050 of Millipore), sonicator (Crest model, USA) and thermostatic water bath (Memmret, Germany) were used. Mass spectrometric analysis is carried out using a TSQ Quantum Access MAX-triple quadruple system. Data acquisition and processing were performed using Thermo Scientific Xcalibur 2.1 software.

2.2. Chemicals and solvents

All chemicals used in the preparation of mobile phase were of HPLC grades, and the reagents were of analytical grades. They included acetonitrile (purchased from Poch, Poland), hydrochloric acid (36%, Fischer Scientific, UK), sodium hydroxide (LOBA Chemie, India), tetra-n-butyl ammonium hydrogen sulphate (Alpha chemika, India) and potassium dihydrogen-o-phosphate (Adwic, Egypt).

2.3. Samples

Imidapril hydrochloride (IMD) reference standard material was kindly supplied by Mitsubishi Tanabe Pharma Factory, its potency was found to be 99.4%. Tanatril tablets were labeled to contain 10 mg of IMD per tablet and manufactured by HIKMA Pharmaceuticals, Amman-Jordan. They were purchased from the local markets.

2.4. Chromatographic conditions

At room temperature, the separation was carried out on ACE Generix 5C8 column (15 cm × 4.6 mm i.d., 5 μm practical size). The mobile phase consisting of buffer (0.1 M potassium dihydrogen-o-phosphate, 0.02 M tetra-n-butyl ammonium hydrogen sulphate) and acetonitrile 60 : 40 (v/v) at pH = 4.5, was adjusted with 1 N phosphoric acid. The prepared mobile phase was filtered through a 0.45 μm filter under vacuum, degassed and sonicated for 5 min. The flow rate was kept at 0.7 mL min⁻¹. The injected volume was 20 μL and the eluent was monitored at 210 nm.

2.5. Preparation of standard solution

A stock standard solution of IMD (2.0 mg mL⁻¹) was prepared in a solvent of water: acetonitrile (60 : 40 v/v) by weighing and transferring 100 mg of IMD into dry and clean 50 mL volumetric flask and completed to the mark with solvent. The prepared solution was further diluted by the mobile phase to get a working standard solution having the concentration of 0.5 mg mL⁻¹.

2.6. Construction of calibration curve

Aliquots of standard drug solutions were transferred from working standard solution into a series of 10 mL volumetric flasks to get concentrations of equivalent to 0.1–100 μg mL⁻¹. Triplicate of 20 μL of each drug solution were injected and different chromatograms were recorded at 210 nm under the previously described chromatographic conditions. Calibration curve was constructed and the regression equation was computed by plotting the peak area against the corresponding concentration in μg mL⁻¹.

2.7. Preparation of alkaline degradation products

Alkaline degradation of IMD was carried out by dissolving about 12.5 mg of IMD in 10 mL 0.1 N NaOH and quantitatively transferred into 50 mL volumetric flask. Complete hydrolysis was achieved at room temperature after 3.5 hours and then neutralized by adjusting the pH to 7.0 with 0.1 N HCl. After neutralization the volume was completed with the solvent to obtain stock solution of IMD degradation products equivalent to 0.25 mg mL⁻¹. This stock solution was diluted with the mobile phase to get solution has a concentration of 25 μg mL⁻¹. The obtained degradation products were subjected to MS spectroscopic analysis for subsequent structural elucidation.

2.8. Analysis of laboratory prepared mixture

For HPLC, laboratory prepared mixtures of IMD and its alkaline degradation products were prepared from the working standard solution and stock solution of degradates and diluted to the mobile phase in the range of 20–180% w/w. The procedure was undertaken as described above where the concentration of IMD was calculated from the corresponding regression equation.

2.9. Assay of IMD in tanatril tablets

Ten tablets of tanatril were finely powdered. A portion of the powdered tablets equivalent to 50 mg of IMD was transferred into 100 mL volumetric flask and sonicated for 20 min with 60 mL water. Then, the volume was completed with acetonitrile and filtered to prepare a stock solution having the concentration 0.5 mg mL⁻¹. Aliquots of 1.0 and 5.0 mL were transferred to 50 mL volumetric flasks and the volume was completed with the mobile phase. Then, 20 mL from each final dilution were injected in triplicate. The general procedure described above was followed. Then, the concentration of IMD in its pharmaceutical preparation was calculated.

2.10. Application of standard addition technique

To check the validity of the proposed chromatographic method, standard addition technique was applied. Known amounts of IMD working standard were added to a pre-quantified sample solution (tanatril), and then the mean recovery% of the pure drug was calculated and a relation between concentration and area was drawn once when standard alone and the other when adding sample.

2.11. Kinetic investigation of the alkaline degradation of IMD

Accurately weighed amount of about 50 mg of IMD was dissolved in 50 mL of solvent. 1 mL aliquots of the above solution were transferred to separate series of test tubes and mixed with 1 mL of 0.1 N NaOH or 0.2 N NaOH (to study the effect of NaOH concentration). The test tubes were stoppered and placed in a thermostatic water bath at different temperatures 16, 25, 40, 55 and 70 °C (to study the effect of temperature) for different time intervals. At the specified time intervals, the contents of the tubes were neutralized to pH 7.0 using 1 mL of 0.1 N HCl. The content of each tube was immediately transferred to 10 mL volumetric flasks and diluted to volume with mobile phase. Aliquots of 20 μL of each solution were chromatographed under the conditions described above. The concentrations of the remaining IMD were calculated at each time interval and temperature. Logarithm of the percentage of the remaining IMD concentration was plotted against the corresponding time interval in hours for each temperature, and the regression equations were computed.

2.12. Studying the effect of oxidizing, acidic and thermal conditions on the stability of IMD

Aliquots of IMD equivalent to 12.5 mg were transferred separately into four series of 50 mL volumetric flasks. To the first flask, 10 mL of 0.1 N HCl were added and the solutions were left for 4 hours then neutralized. To the second and third flasks, 10 mL of 3% H₂O₂ and 10% H₂O₂, respectively, were added and the solutions were left for 24 hours. The last flask was placed in an electrical oven with 80 °C for 24 hours. The flasks were completed to volume with the solvent in all cases. After the mentioned time, 20 μL of the solutions were injected into the liquid chromatograph using the chromatographic conditions which described above.

3. Results and discussion

3.1. Identification of the degradation products

IMD was left with 0.1 N sodium hydroxide at room temperature for 3.5 hours. Complete degradation of IMD drug gives four degradation products (DI-IV) which analyzed and separated by HPLC at retention times of 3.92, 4.15, 4.45 and 4.92 ± 0.15 min for DI, DII, DIII and DIV, respectively, with the disappearance of the peak of intact drug at retention time of 8.20 ± 0.15 min (Fig. 2A and B).

Fig. 2 HPLC chromatograms of (A) imidapril HCl, (B) complete alkaline degradation (3.5 hours) and (C) complete alkaline degradation after 5 hours.

It is noticed that if IMD was left with 0.1 N NaOH at room temperature more than 3.5 hours, the peak of DIV with retention time 4.92 min decreased and at 5 hours it disappeared (Fig. 2C).

The identification of IMD degradation products was performed by means of LC-MS method. From MS spectrum, the ion peak of IMD was identified at m/z 406 (Fig. 3A). While those of alkaline degradation products were found at m/z 378, m/z 280, m/z 252 and m/z 208 as shown in (Fig. 3B).

Fig. 3 Mass spectra of (A) imidapril HCl and (B) complete alkaline degradation products.

The suggested pathways for the degradation of IMD in 0.1 N NaOH are presented in Scheme 1, which explain that the formation of m/z 378 corresponded to a diacidic derivative of IMD formed by its ester bond hydrolysis. While the medium of degradation solution is full of OH⁻ ions which easily make nucleophilic substitution to break amide and replace N with OH⁻ ion giving the parent acid degradation product at m/z 280. And the same nucleophilic substitution and the breaking of amide is occurred for m/z 378 to form the product at m/z 252 from m/z 378 not m/z 406 which may explain the disappearance the peak of DIV with increasing the time of the reaction. This means that DIV is imidaprilat at m/z 378 (ref. 15) and finally m/z 208 is formed easily via bond cleavage bond between CH₃–C–NH as shown in Scheme 1.

Scheme 1 The proposed pathway of alkaline degradation of imidapril HCl.

3.2. Method development and optimization

To optimize the HPLC assay parameters, the type of column used, its dimensions, its mobile phase conditions, and its choice of detection wavelength were carefully investigated. Different types of stationary phases, columns with different dimensions, and particle sizes were tried. It was found that the ACE Generix 5C8 column (250 × 4.6 mm) with a particle size of 5 μm gave suitable resolution for IMD and its degradation products (DI-IV). The mobile phase was chosen after several trials to reach the optimum stationary/mobile phase matching to achieve the best resolution and optimum system suitability parameters according to USP.¹⁹ Several mobile phase compositions with different buffer of different pH values were employed. The results of these trials showed best results using buffer consisting of 0.1 M potassium di-hydrogen-ortho-phosphate, 0.02 M tetra-n-butyl ammonium hydrogen sulphate (pH = 4.5 with 1 N NaOH) and acetonitrile in ratio 60 : 40 (v/v). The flow rate was isocratic adjusted at 0.7 mL min⁻¹ as shown in (Fig. 4).

Fig. 4 Chromatogram of laboratory prepared mixture of imidapril HCl and its degradation products.

The effect of pH on some system suitability parameters; including retention time, and resolution, was carefully studied using the HPLC system as shown in (Fig. 5A and B).

Fig. 5 Effect of pH on (A) retention time and (B) resolution of imidapril HCl and its degradation products using the proposed HPLC method.

The retention time of the basic drug IMD and DIV (imidaprilat) decreased with decreasing pH which became 4.59 min at pH 2 for both substances. This is probably due to the effect of ion pairing with the ionized drug. While the retention times of DI, DII and DIII were nearly constant overall pH range as shown in (Fig. 5A). While from (Fig. 5B), it is showed that the best resolutions between the basic drug and the four degradation products appeared from pH 3 to 5, so the best pH for the method was at 4.5.

3.3. System suitability test

System suitability testing parameters were calculated according to USP to ensure that the chromatographic systems were working correctly during the analysis after optimizing the chromatographic conditions using different mobile phases, pH, and flow rates. Selectivity factor (α), resolution (R), column efficiency, tailing factor (T) and relative standard deviation peak area of ten replicate injections were parameters to be checked during the analysis as represented in Table 1.

Table 1 System suitability test results of the proposed HPLC method for the determination of imidapril HCl

Parameter	Imidapril HCl	DIV	DIII	DII	DI	Reference values
Retention time (min)	8.194	4.962	4.464	4.175	3.935	—
Selectivity (α)	3.31	1.55	1.47	1.64	—	α > 1
Resolution (R)	—	16.07	18.46	19.71	23.34	R > 2
Tailing factor (T)	1.278	1.117	1.237	1.215	1.258	T ≤ 2
RSD% of peak areas	0.196	0.824	0.135	0.562	0.528	<1, n = 10
Theoretical plates (N)	16 893	17 704	13 974	11 706	19 733	>2000

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3.4. Method validation

Validation is an act of proving that any procedure, process, equipment, material, activity or system performs as expected under given set of conditions and also give the required accuracy, precision, sensitivity, ruggedness, etc.²⁰ Therefore, validation of the method was performed in accordance to ICH guidelines.

3.4.1 Linearity and range. Calibration curve was constructed representing the relationship between integrated peak areas and the corresponding concentrations in the range of 0.1–100 μg mL⁻¹ as shown in Fig. 6. The characteristic parameters for the regression equation were computed as illustrated in Table 2.

Fig. 6 Calibration curve for imidapril HCl (0.1–100) μg mL⁻¹.

Table 2 Linearity parameters of the proposed method

Parameters	Imidapril HCl
Linearity range, μg mL^−1	0.1–100
LOD, μg mL^−1	0.0069
LOQ, μg mL^−1	0.0211
Regression equation	y = 61.41x + 8.303
Slope	61.41
SE of slope	1.95878 × 10^−5
Intercept	8.303
SE of intercept	0.05962351
Regression coefficient (r^2)	0.99999
SE of estimation	0.129459206

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3.4.2 Precision. The repeatability of the proposed method was evaluated by assaying three samples solutions of IMD within the same day and under the same experimental conditions (intra-day). The precision was evaluated by assaying solutions on three consecutive days (inter-day). Peak areas were determined and the precision was expressed as %RSD < 2. From the data obtained in Table 3, the developed RP-HPLC method was found to be precise.

Table 3 Precision data for the proposed method^b

Drug	Concentration (μg mL^−1)	Intra-day precision		%Mean recovery ± SD	Mean %RSD
		Amount found^a	%Recovery^a
a Mean of three different samples for each concentration.b (n) mean of three different samples for each concentration.
Imidapril HCl	8.00	8.02	100.25	99.65 ± 0.53	0.53
	20.0	19.86	99.29
	60.0	59.64	99.40

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Drug	Concentration (μ mL^−1)	Inter-day precision		Mean %Recovery ± SD	Mean %RSD
		Amount found (n)	%Recovery (n)
Imidapril HCl	8.00	7.99	99.86	99.55 ± 0.29	0.29
	20.0	19.90	99.50
	60.0	59.57	99.28

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3.4.3 Limits of detections and quantifications. The detection and quantification limits were calculated based on the standard deviation of the response and the slope of the calibration curve, as follows:

LOD = 3.3 × σ/S, (1)

LOQ = 10 × σ/S, (2)

where σ is the standard deviation of the response and S is the slope of the regression line of the calibration curve, indicating the sensitivity of the method as shown in Table 2.

3.4.4 Specificity. The specificity of a method is the extent to which it can be used for analysis of a particular analyte in a mixture or matrix without interferences from other components. The specificity of the proposed method was tested by spiking the IMD with appropriate levels of degradation products. The specificity was demonstrated by the chromatograms recorded for mixtures of IMD and its degradants (Fig. 4), indicating that the method enabled highly specific analysis of the drug. Satisfactory results were obtained (Table 4) indicating that high specificity of the proposed method for determination of IMD in presence of up to 90% of its degradation products.

Table 4 Determination of IMD in presence of its degradation products in laboratory prepared mixtures by the proposed stability-indicating chromatographic method

Drug%/degradation products% (w/w)	Recovery%	Mean %Recovery ± SD
100/20	99.01	99.47 ± 0.54
100/60	99.27
100/100	99.29
100/140	99.40
100/180	100.4

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3.4.5 Peak purity. The selectivity of the proposed method was further demonstrated by testing peak purity of the cited drug in pharmaceutical preparation matrix spiked with its degradation products using diode array detector (DAD) as illustrated in (Fig. 7).

Fig. 7 Peak purity of IMD in drug product matrix spiked with its degradation products.

The main feature of DAD is the possible collection of spectra across a range of wavelengths at each data point collected across a peak, and through software manipulation where each spectrum can be compared to determine peak purity.²⁰ Comparing peak spectra is probably the most popular method to discover an impurity. If a peak is pure all UV-visible spectra acquired during the peak's elution or migration should be very close to a perfect 100% match.²¹ The similarity factor of IMD peak was found to be 999.933 indicating that the spectra are very similar (similarity factor > 995) and hence drug is pure.

3.4.6 Accuracy. The interference of excipients in the pharmaceutical formulations was studied using the proposed method. For this reason, standard addition technique was applied to the commercial pharmaceutical formulation containing IMD (tanatril tablets). In this application, the mean percentage recoveries and their standard deviation for the proposed method were calculated (Table 5 and Fig. 8). According to the obtained results, a good precision and accuracy was observed for this method. The excipients in pharmaceutical formulations do not interfere in the analysis of IMD in its pharmaceutical formulation.

Table 5 Standard addition data of IMD for the proposed method

Drug	Label claim/tablet (μg mL^−1)	Amount of standard added (μg mL^−1)	Amount found (μg mL^−1)	%Recovery^a	%Mean recovery ± SD	Mean %RSD
a Mean of three different samples for each concentration.
Imidapril HCl	10	2.5	2.476	99.87	99.66	0.64
		5	5.004	100.6
		7.5	7.447	99.07
		10	9.907	99.29	±0.64
		15	15.09	100.1
		20	19.97	99.04

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Fig. 8 Standard addition plots of IMD drug.

The results obtained by applying the proposed chromatographic method were statistically compared to those of the reported HPLC method¹⁴ used for IMD analysis. It is concluded that with 95% confidence, there is no significant difference between them since the calculated t- and F-values are less than the theoretical values; as presented in Table 6.

Table 6 Statistical analysis of data obtained for the determination of IMD drug^a

Parameters	Proposed method	Reported method
a Tabulated F and t values at 95% confidence limit: 6.39 and 2.77, respectively.
Mean	100.0683	99.925
SD	0.4502	0.5067
Variance	0.202657	0.25679
SE	0.1838	0.2069
t-Test	0.517971	—
F-Test	0.789192	—

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3.4.7 Robustness. Robustness was performed by deliberately changing the chromatographic conditions. The flow rate of the mobile phase was changed from 0.7 mL min⁻¹ to 0.6 mL min⁻¹ and 0.8 mL min⁻¹. The organic strength was varied, as the acetonitrile proportion from 40% to 41% and 39%. The buffer pH value was changed from pH 4.5 to pH 4.4 or pH 4.6. Only one factor was changed at a time, while the others were kept constant. These variations did not have significant effect on chromatographic resolution by the proposed LC method for IMD and their alkaline degradation products, indicating good robustness of the proposed LC method (Table 7).

Table 7 System suitability parameters and robustness in normal and changed condition

System suitability parameters	Resolution	Retention time	Theoretical plates	Tailing factor	Selectivity
Robustness
0.6	16.80	9.218	18 108	1.304	3.30
Flow rate 0.7	16.07	8.194	16 893	1.278	3.31
0.8	15.35	7.170	15 225	1.263	3.31
4.4	15.55	8.111	16 981	1.232	3.23
Buffer pH 4.5	16.07	8.194	16 893	1.278	3.31
4.6	16.93	8.606	16 484	1.324	3.44
59 : 41	18.72	8.931	16 877	1.262	3.38
Buffer : ACE 60 : 40	16.07	8.194	16 893	1.278	3.31
61 : 39	18.99	9.177	17 189	1.302	3.37

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3.5. Stability study

Stability study of IMD drug under different stress degradation process such as acid, an oxidizing agent namely hydrogen peroxide and finally exposure to electrical oven (80 °C) for a fixed period of time (as mentioned in the experimental part) resulted in nearly no change in the chromatograms of the drug, as shown in Fig. 9A–E.

Fig. 9 Chromatograms representing (A) IMD standard, (B) result of acid induced degradation, (C) thermal induced degradation, (D) and (E) oxidizing agent induced degradation for 10% and 3% H₂O₂, respectively.

3.6. Kinetic study

The kinetics of alkaline degradation of IMD was investigated using 0.1 N NaOH, since the decomposition rate was convenient to obtain reliable kinetic data. When degradation process was induced and monitored using the proposed HPLC method, regular decrease in the concentration of the drug with increasing time intervals was observed. Where the logarithm percentage of the remaining concentration for the drug was plotted against the corresponding time interval in hours where straight line was obtained indicating first-order degradation (Fig. 10). Since the hydrolysis was performed in a large excess of NaOH (0.1 N), it followed a pseudo first-order reaction rate. The term is used when two reactants are involved in the reaction, but one of them is in such a large excess (NaOH) that any change in the concentration is negligible compared with the change in the concentration of the other reactant (drug).

Fig. 10 First order plot of hydrolysis of IMD with 0.1 N NaOH.

Different parameters that affect the rate of the reaction were studied. The temperature dependence and effect of base strength on IMD degradation was studied by conducting the reaction at different temperatures (16, 25, 40, 55 and 70 °C) using different base strengths (0.1 and 0.2 N NaOH) (Fig. 11A and B).

From the slopes of the regression lines, it was possible to calculate the apparent first order degradation rate constant (K_obs) and the half-life at each temperature in Table 8 according to the following equations

log(C_t/C_o) + 2 = −K_obst, (3)

t_1/2 = 0.693/K_obs (4)

where C_t = concentration remaining at time t, C_o = initial concentration, K_obs = apparent rate constant, t_1/2 is the half life.

Fig. 11 First order plot of the hydrolysis of IMD with (A) 0.1 N NaOH and (B) 0.2 N NaOH at different temperatures.

At each temperature, the rate constant and t_1/2 were calculated. It was concluded that as the temperature increased, the rate of hydrolysis also increased, with a noticed decreased in the t_1/2 as shown in Table 8.

Table 8 Kinetic data of the stress alkaline hydrolysis of IMD using the proposed HPLC method

Strength of NaOH	Temperature (°C)	Kobs (h^−1)	Half life t1/2 (h)
0.1 N	16	0.130	5.330769
	25	0.203	3.413793
	40	0.497	1.394366
	55	1.223	0.566639
	70	2.169	0.319502
0.2 N	16	0.328	2.112805
	25	0.430	1.611628
	40	0.95	0.729474
	55	2.245	0.308686
	70	3.433	0.201864

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By plotting log K_obs values versus 1/T (inverse absolute temperature), the Arrhenius plots were obtained as shown in (Fig. 12).

Fig. 12 Arrhenius plots of alkaline degradation process with different strength of NaOH.

The activation energy was calculated by applying the following equation:

log K₂/K₁ = E_a/2.303R × T₂ − T₁/T₂T₁ (5)

where E_a is the activation energy, T₁ and T₂ are the two temperatures in Kelvin, R is the gas constant, and K₁ and K₂ are the rate constants at the two temperatures used. The calculated E_a was found to be 42.023 K joule mol⁻¹ and 21.541 K joule mol⁻¹ for IMD at 0.1 and 0.2 N NaOH, respectively.

From the previous study it was found that increasing the base strength results in a uniform increase in the rate constant and a decrease in the t_1/2, revealing high instability of the drug towards higher base strengths that was also confirmed by its law value of the activation energy.

4. Conclusion

The proposed HPLC method provides simple, sensitive, and specific method suitable for the quantitative analysis of IMD drug in the presence of its alkaline degradation products for the routine quality control analysis either in its pure form or in available pharmaceutical dosage form with no interference from the excipients or the degradation product. The reaction kinetic of the degradation was found to be a pseudo first order reaction under the experiment's basic stress conditions. Other stress conditions did not affect the drugs investigated. The methods proved that the selectivity, accuracy, and simple mobile phases used provide simple and economic applications, and they reflect suitability for quality control laboratories applications. The proposed method could be for extended for estimation studies in human plasma.

Acknowledgements

The authors are thankful to Laboratory of HPLC in NODCAR-Egypt for providing facilities for research work.

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