Introduction
Diarrhea is defined as three or more soft or watery stools per day (or more than a normal defecation habit). Acute gastroenteritis is defined as softening of stool consistency and/or increased frequency (3 or more per day). Cholera is a pathogen in the group of bacterial gastroenteritis and causes acute diarrhea without causing intestinal damage (1). Thiorphan is an inhibitor of cell membrane peptidase enzyme enkephalinase, which is found in various tissues, especially in the epithelium of the small intestine. This enzyme ensures the digestion of exogenous peptidase and degradation of endogenous peptidase such as enkephalin. Racecadotril (acetorphan) is a prodrug that hydrolyzes to its active metabolite, thiorphan (Figure 1) (2). Racecadotril protects enkephalins from enzymatic degradation. Thus, it prolongs its effect on enkephalinergic synapses in the small intestine and reduces hypersecretion. Racecadotril is a pure intestinal antisecretory drug. It reduces water and electrolyte loss caused by cholera toxin or inflammation and does not affect basal secretory activity. Racecadotril rapidly performs its antidiarrheal effect without changing the duration of intestinal transit (3).
When the analyzes of racecadotril in pharmaceutical preparations were examined, it was found to be analyzed by spectrophotometric (4,5), HPTLC (6), and HPLC (7-14). In this study, it was planned to develop a new method for the analysis of racecadotril in tablets, using ultra-performance liquid chromatography (UFLC) method, which was more sensitive, reproducible and had a shorter analysis time than high-performance liquid chromatography (HPLC). This proposed method was also validated and its applicability to analyzes in pharmaceutical preparations was tested.
Methods
Chemicals and Solvents
The racecadotril substance was obtained from Ilko Pharmaceuticals. The pharmaceutical preparation was purchased from the pharmacy (raxerin 10 mg & 20 mg Tablet).
Acetonitrile: Merck (Acetonitrile gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur. Acetonitrile CAS 75-05-08, molar mass 41.05 g/mol, and chemical formula CH3CN.)
Racecadotril Standard Solution
Stock solution: Twenty-five mg of racecadotril was weighed exactly and transferred to a 25 mL flask, 10 mL of dilution solution (as mobile phase) was added and dissolved with the help of vortex, and its volume was completed with the mobile phase (equivalent to 1 mg/mL racecadotril). Racecadotril stock solution is stable for approximately 29 days at +40 oC.
Tablet Sample Solutions
80.0 mg of raxerin 10 mg dispersible tablet and raxerin 30 mg dispersible tablet powders were weighed separately, transferred to a 100 mL flask, 50 mL of mobile phase was added, dissolved in an ultrasonic bath for about 30 minutes and allowed to cool, the volume was completed with mobile phase (0.1 mg/mL). Stock solution is stable for approximately 29 days at +40 oC, 7 days at room conditions.
Chromatographic System
UFLC Device (Shimadzu Nexera X2, Binary Pump)
LC 20AB pump system, SPD M20A Detector (Photo Diode Array), LC Solution System software, SIL 20AC Autosampler, CTO-10As Column furnace, Analytical column: Inertsustain C18 (4.0x100 mm, 3 µm; GL Sciences, Tokyo- JAPAN). Mobile phase is consisted of 70% ultra pure water & 30% acetonitrile
Method Validation
After method development was completed, analytical method validation was performed according to ICH guideline Q2(R1) (15).
Acuracy
The standard addition method was used to determine the accuracy of the developed method. For this purpose, 40 mg of tablet powder in two different forms (10 mg-30 mg) was weighed separately and transferred to a 10 mL balloon. 2, 5, 10 µg/mL concentrations of racecadotril standard solutions were added onto the sample solutions at a concentration of 5 µg/mL and completed to 10 mL with mobile phase (dilution solution).
Inter-day and Intra-day Reproducibility
In order to determine the precision of the method, a reproducibility study was carried out for 6 repetitions for 3 different days for solutions with active substance content at a concentration of 20 µg/mL.
Lineer Range
In order to show this linearity range, solutions containing racecodotril at 5 different concentrations were prepared. Peak areas and concentration versus linearity plots obtained from the chromatograms were plotted.
Robustness study
Three parameters were determined for the robustness test of the method. (Column temperature change (±2 ºC), Filter effect (PTFE, PVDF, Nylon), Flow rate change (±0.1 mL/min). The applicability of the analysis method in these different conditions was tested.
Solution Stability
In order to determine the racecadotril solution stability, standard solutions and sample solutions were kept at room temperature for 7 days and at +4 oC for 29 days.
Degredation process
In this study, standard solutions with a concentration of 20 µg/mL were prepared. These solutions were subjected to temperature, oxidation, acidic and basic stress conditions and analyzed.
Analysis of Racecadotril in Pharmaceutical Preparations
Analysis with the Developed Method
In order to see the applicability of this developed method in tablets, 80 mg of the powder of the tablets in two different forms (10 mg and 30 mg) was weighed and transferred into a 10 mL flask. Adding 50 mL of mobile phase, it was dissolved in an ultrasonic bath for about 30 minutes, allowed to cool, and completed to its volume with mobile phase (0.1 mg/mL). Two mL of this prepared stock solution was taken and transferred to a 10 mL balloon flask, and its volume was completed with mobile phase (20 µg/mL).
Analysis by Comparison Method
The racecadotril method in the Pharmacopeia was determined as the comparison method (16). By following the sample preparation conditions in the Pharmacopoeia method, separate solutions were prepared for 10 mg and 30 mg tablets with a concentration of 20 µg/mL.
Results
Determination of Chromatographic Conditions
As a result of the experiments, the best results were obtained in a C18 column with a length of 10 cm, an inner diameter of 4.0 mm, a particle diameter of 3 µm, acetonitrile-water (70:30) mobile phase system, at 30 oC, at a flow rate of 0.7 mL/min. The PDA detector was studied in the range of 190-300 nm, and the most sensitive results were obtained at a wavelength of 220 nm. The retention time in this chromatographic system is 2.56 minutes for racecadotril. In Figure 2, the standard chromatogram of racecadotril at 220 nm wavelength is indicated.
Accuracy
The standard addition method was used to determine the accuracy of the developed method.
The data obtained as a result of the analysis and the recovery percentages were calculated from the formula (Ct-C1 ) x100/C2.
Ct: Total concentration of racecadotril found,
C1: Analyte concentration from the pharmaceutical preparation,
C2 : The concentration of the added standard solution.
Recovery results were found in the range of 98.28-101.17% (Table 1). The results obtained proved that the additives in the tablets did not interfere.
Inter-day and Intra-day Reprocubility
Relative standard deviation (RSD) values of the analyzes performed on the same day, with 6 repetitions on three different days, were calculated between 0.25-0.40. The intraday standard and RSD between tablets were calculated as 0.57% (Table 2). The RSD values obtained as a result of the analyzes performed on different days were calculated between 0.47 and 0.49. The RSD between the standard and tablets for the different day was calculated as 0.35% (Table 2). The results obtained were the analysis values for three consecutive days.
Lineer Range
For racecadotril, 5 different concentrations of standard solutions were studied in the concentration range of 5-100 µg/mL. The line equation was found to be y=10362x+9666.4 r2=0.9989.
Robustness study
Different conditions were applied to determine the robustness of the developed method. As a result of the analysis, the retention time at 0.60 mL/min flow for the flow test was 2.98 min; it was observed as 2.25 min for 0.80 mL/min and 2.55 min for normal conditions. The data obtained are shown in Table 3.
Solution Stability
To determine the solution stability, standard and sample solutions were kept in the dark for 7 days at room temperature, 24 hours and 48 hours in the automatic sample sampler, and 29 days at +4.00 oC. As a result of the evaluations, it was determined that the solutions remained stable. There was no deviation of more than 1.00% within 48 hours.
Degradation Procedure
Stress studies were carried out to show the effect of the related stress conditions in the developed method. The results are shown in Table 4. Chromatograms are shown in Figure 3.
Analysis of Racecadotril with the Method Developed in Pharmaceutical Preparations
It was studied to see the applicability of the developed method on tablets. The amount of racecadotril obtained in tablet samples was calculated using the measurement curve equation. In addition, the comparison method was also studied (16). The results obtained and the t- and F-test values are shown in Table 5.
Discussion
In this study, a new method was developed for the analysis of racecadotril in tablets, using UFLC method, which was more sensitive, reproducible and had a shorter analysis time than HPLC. The UFLC method offers benefits as an alternative to conventional HPLC. It does not only reduce the period required to complete the analyses and conserves the solvent used for the process, but it also allows for separating and determining the drug substance reliably in pharmaceutical formulation. These advantages of the UFLC method provides increased efficiency of the analysis (17). In addition to the developed methods, a degradation study was also carried out. In this study; acid, base, peroxide decomposition and heat decomposition processes were applied. When the results obtained were examined, it was observed that racecadotril was slightly affected by acid, peroxide and heat, but completely decomposed in the base environment. This developed and validated method is reliable, fast, and can be used routinely in pharmaceutical analysis and quantification.
Ethics
Ethics Committee Approval: Since we do not work with biological materials in our study, ethical committee approval is not necessary.
Peer-review: Externally peer reviewed.
Authorship Contributions
Surgical and Medical Practices: E.B., Concept: A.Ö., E.B., Design: A.Ö., E.B., Data Collection or Processing: A.Ö., E.B., Analysis or Interpretation: A.Ö., E.B., Literature Search: E.B., Writing: A.Ö., E.B.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.