Stability-Indicating HPLC Determination of Trandolapril in Bulk Drug and Pharmaceutical Dosage Forms

A rapid, simple, accurate, precise, economical, robust, and stability indicating reverse phase HPLC-PDA procedure has been developed and validated for the determination of trandolapril. The trandolapril was separated isocratically on Hypersil-Gold C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase consisting of 50% acetonitrile and 50% water (containing 0.025% triethylamine, pH 3.0 ± 0.1), at 25 ± 2°C. Retention time of the drug was ~4.6 min. The eluted compounds were monitored and identified at 210 nm. The linearity of the method was excellent (r 2 > 0.9999) over the concentration range of 1–24 μg/mL; the limit of detection (LOD) and limit of quantitation (LOQ) were 0.0566 μg/mL and 0.1715 μg/mL, respectively. The overall precision was less than 2%. Mean recovery of trandolapril was more than 99%; no interference was found from the component present in the preparation. Stability studies indicate that the drug was stable to sunlight and UV light. The drug gives 6 different oxidative products on exposure to hydrogen peroxide. Slight degradation was observed in acidic condition. Degradation was higher in the alkaline condition compared to other conditions. The robustness of the method was studied using factorial design experiment.


Introduction
Trandolapril ( Figure 1) is a long-lasting angiotensin converting enzyme (ACE) inhibitor which was approved by the U.S. Food and Drug Administration for lowering blood pressure in doses up to 2 mg even after discontinuation of treatment [1]. It is also used for patient with evidence of Left Ventricular (LV) systolic dysfunction after Acute Myocardial Infarction (AMI). It can be given safely over a prolonged period of time [2]. Trandolapril is the international nonproprietary name of (2S, 3aR, 7aS)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino}propanoyl]-octahydro-1H-indole-2-carboxylic acid. Trandolapril is rapidly absorbed and metabolized to its biologically active diacid form, trandolaprilate, in liver which shows high lipophilicity compared to other ACE inhibitors. Determination of trandolapril alone has been analyzed less often than other drugs; indeed, only few methods involving amperometric biosensors [3], a potentiometric enantioselective membrane electrode [4], liquid chromatography tandem mass spectrometric [5], HPLC [6][7][8][9][10][11][12][13], HPTLC [14,15], UV spectroscopy [16], and capillary electrophoresis [17] have been used. Trandolapril is official in British Pharmacopoeia and it is estimated by nonaqueous titration [18]. A few stability-indicating HPLC methods [11][12][13] have been reported, which provides variable level of degradation of trandolapril. Stability-indicating method reported by Manju Latha and Gowri Sankar [11] does not produce any degraded product in different stressed conditions, although it is well documented that trandolapril is susceptible to hydrolysis. Impurity profiles of trandolapril under stress (acidic and neutral) conditions studied by Dendeni et al. [12] require sophisticated LC-MS-MS instrument. Sahu et al. [13] have reported the validated stability-indicating method which can separate the hydrolytic degraded product of trandolapril. However none of the HPLC method reported the oxidative degraded product of trandolapril. International Conference on Harmonization (ICH) guidelines [19] require performance of stress tests on the drug substance, which can help to identify the likely degradation products. Moreover, validated stability-indicating methods should be applied in the stability studies [20] once they have demonstrated their suitability for their intended purpose. Thus, stability-indicating methods have to demonstrate that they are specific, which involves evaluating the drug in the presence of its degradation products. The aim of the present work was the development and validation of an HPLC stability-indicating method for determining trandolapril in its pharmaceutical form following ICH recommendations to achieve this goal; a stress study of the drug was performed in order to validate the stabilityindicating power of the developed analytical method and to identify the key factors that will impact the stability of the drug product. The robustness of the developed method was studied using design of experiment utilising factorial design.  [19,20]. A 5% significance level was used for evaluation. LOD and LOQ were determined from the calibration function.

System Suitability.
System suitability parameters were tested with six replicate injections of the diluted sample of working standards (10 g/mL) at the start of the project and at the end of the project. The system suitability parameters were calculated using the internal features of LC-Solution software as per United States Pharmacopoeia [21]. The parameters were retention time, peak area and height, width at half peak height, tailing factor, efficiency, and height equivalent theoretical plate (HETP). System suitability was measured on the basis of precision (RSD). The precision, as measured by coefficient of variation was determined at each set's parameters and it should be less than 2% at the beginning of validation and at the end of validation.

Precision.
Precision was measured using triplicate determination of quality control samples of 4 g/mL (LQC), 12 g/mL (MQC), and 22 g/mL (HQC) of trandolapril, on three different occasions (0, 3, and 6 h) and different days. The precision (RSD) of the method was determined as intraday precision (repeatability) and intermediate precision.
The intermediate precision was estimated from the RSD of the analysis of the samples prepared at the same concentration but on 3 different days at different concentration levels, while intraday precision was calculated by analyzing the same concentration during the same day at different time.

2.4.4.
Accuracy. Accuracy (as percentage recovery) was measured using replicate sample of trandolapril prepared using capsule matrix. Different samples ( = 3, at each level of 80%, 100%, and 120%) were prepared using capsules content (2 mg as 100%) and adding known quantity of trandolapril (at 80%-120% level). From these fortified samples, appropriate sample solutions were prepared and analyzed and the total amount recovered was calculated. Accuracy was calculated by comparing with true value. The concentrations were back-calculated by regression equation = 21225 + 1303 (weighting factor ).

Robustness.
It is a measure of reproducibility of test results under normal, expected, operational condition from analyst to analyst. The robustness of the method was evaluated on the basis of precision, as measured by percent coefficient of variation (% CV or RSD), determined as each concentration level was required not to exceed 2%. Design of experiments (DOE) was used to study robustness of the method. A 2 4 factorial design was used to test the robustness of chromatographic separation. The experimental design is useful for this kind of study as it facilitates the investigation of several parameters by reducing the number of experiments. Acetonitrile content of the mobile phase, volume of peak modifier, pH, and flow rate were investigated. Upper and lower limits are shown in Table 5. The experiments were run randomly and the selected responses were retention time ( ), tailing factor ( ), and area count.
2.4.6. Stability Studies. Stress study like oxidative stress, alkaline stress, acidic stress, exposure to sunlight, and UV light (254 nm) were carried out using trandolapril raw material. Chromatograms were recorded in order to study the specificity of the method. The chromatograms of the samples were compared with those of control samples that were freshly prepared from the stock standard solution and without stress. All samples were analyzed in triplicate. The peak purity was checked using the tools of the LC-Solution software. This assessment was based on the comparison of spectra recorded during the elution of the peak. UV spectra and peak purity were used to assess purity of trandolapril.
(1) Oxidative Stress. Trandolapril (5 mg) was weighed accurately and transferred to 50 mL volumetric flask. 5 mL of 30% hydrogen peroxide was added to it. It was stirred for one hour, and then the contents were diluted to 50 mL with mobile phase. Replicate solutions of concentration 20 g/mL were prepared and chromatograms of these solutions were recorded and compared with the chromatograms obtained from the fresh solution of trandolapril having the same concentration and the chromatogram of the blank (solution containing only hydrogen peroxide).
(2) Effect of Acid and Alkaline Media. Trandolapril (5 mg) was weighed accurately and transferred to 50 mL volumetric flask. It was shaken for one hour with 5 mL of either 0.1 M hydrochloric acid (HCl) or 0.1 M sodium hydroxide (NaOH). After one hour the content was diluted to 50 mL with mobile phase. Replicate solutions of concentration 20 g/mL were prepared and chromatograms of these solutions were recorded and compared with the chromatograms obtained from the fresh solution of trandolapril having the same concentration and the chromatogram of the blank.
(3) Effect of UV Light or Sunlight. Trandolapril API (100 mg) was placed in an open watch glass and exposed to either UV-irradiation (∼100 W/m 2 ) or direct sunlight for two hours with occasionally shifting the content using stainless steel spatula. After two hours 10 mg of trandolapril was weighed and transferred to 10 mL volumetric flask. It was dissolved in mobile phase. 1 mL of the prepared solution was transferred to a 50 mL volumetric flask and diluted to the mark with mobile phase. Chromatograms were recorded and compared with the chromatogram of unexposed API.

Analytical Method Development.
The HPLC method was developed as a stability-indicating method to determine trandolapril in the presence of the possible degradation products (trandolaprilate) of the drug. Therefore, a retention time between 4 and 5 min was chosen for the drug since it allowed both a rapid determination of the drug, which is important for routine analysis, and a complete drug separation. During development step various mobile phases of water : methanol or water : acetonitrile (35 : 65, 40 : 60, 45 : 55, 50 : 50, 55 : 45, 60 : 40, and 65 : 35 v/v, with or without peak modifier, pH 3, 5, or 7) were tried and the responses were recorded. On the basis of responses and chromatographic parameters studied a mobile phase of acetonitrile: water (50 : 50 v/v, containing 0.25 mL/L TEA, final pH adjusted to 3.0 with OPA) was selected as suitable mobile phase, which can separate the drug and degraded products. Under these conditions the drug was eluted at ∼4.3 min at ambient temperature (25 ± 2 ∘ C). The absorption maximum of the drug ( 210 nm) was selected for detection, as there was no interference from excipients present in drug. Figure 2 depicts the representative chromatogram obtained with the present method.

LOD and LOQ.
The LOD and LOQ values were 0.0566 and 0.1716 g/mL calculated using calibration curve as per    [13] were 0.1 and 0.8 g/mL, based on signal to noise ratio method.

Accuracy and Precision.
The accuracy and precision of the analytical method were established across its linear range as indicated in the guideline. As shown from the data in Table 2, excellent recoveries (99.3 to 100.2%) were obtained at different added concentration level. The results obtained for the intraday and interday precision of the method were expressed as RSD values. As shown in the table, the intraday and interday RSD was < 2.0% for all concentrations tested in different situations studied (Table 3).

Specificity.
Specificity of the method was assessed by comparing the chromatograms obtained from capsule content and drug standards. The retention times of drug from standard solutions and from capsule content were identical and no coeluting peaks from the diluents were observed, indicating specific method for quantitative estimation of drug in the commercial formulation.
International Journal of Analytical Chemistry 5 Values represent mean and SD of six determinations.

System Suitability.
System suitability parameters were studied with six replicates standard solution of the drug and the calculated parameters are within the acceptance criteria. The tailing factor, the number of theoretical plates, and HETP were in the acceptable limits (RSD less than 2%). The system suitability results are shown in Table 4.
3.2.6. Robustness. Robustness of the methods was illustrated by getting the resolution factor and tailing factor, when mobile phase acetonitrile content (±5%), pH (±0.25 units), peak modifier (±0.05 mL/L), and flow rate (±0.1 mL/min) were deliberately varied. It was studied using factorial design experiment using Design Expert software version 8.0 (Stat Ease Inc, USA). The deliberate changes in the method do not affect the retention time, tailing factor, and area count for drug significantly. The scaled and centered coefficient plots for the above responses revealed that different parameters did not affect responses significantly, so that the developed method was considered rugged and robust. Results are presented in Figure 3 and Table 5.  Table 6). Figure 4(b) represents the chromatogram, peak purity, and UV spectra of the freshly prepared sample. After exposure to 0.1 M NaOH  (Figure 4(c)). Results also indicate trandolapril degrades after exposure to 0.1 M HCl and forms degradation product having retention time 3.920 and 6.129 min. The percentage of undegraded trandolapril was 99.6% (Figure 4(d)). No degradation products were produced on exposure to the UV light or sunlight, which indicates that trandolapril has high stability under these stressed conditions. LC-MS-MS identification of different degraded product and impurity profile of trandolapril under acidic and neutral conditions has been reported [12]. Sahu et al. [13] studied the hydrolytic decomposition of trandolapril (at a drug concentration of 2 mg/mL) under different conditions (acidic, alkaline, or neutral) at 80 ∘ C. The degradation of trandolapril was 50 and 65% under acidic and alkaline conditions, respectively. None of these studies (HPLC) reported the oxidative degradation of trandolapril. Our results reveal that the trandolapril is also susceptible to the oxidation. Vikas et al. [15] reported the two oxidative products of trandolapril using developed and validated HPTLC method.
3.2.8. Assay. The proposed method was applied to the determination trandolapril in capsule formulations. The results of these assays yielded 99.2% (RSD = 0.89%) of label claimed.  Low value of precision indicates that the method can be used precisely for the estimation of drug in formulations (Table 7).
International Journal of Analytical Chemistry

Conclusion
A validated HPLC method has been developed for determination of trandolapril in formulations. The proposed method is simple, economical, accurate, precise, specific, robust, and stability-indicating. Robustness of chromatographic method was studied using design of experiments indicating robust and rugged method of analysis can be easily and conveniently adopted for the routine analysis of trandolapril in pharmaceutical dosage form and bulk drug.