The necessary products for preparation of the mobile phase and used buffers are of analytical grade, whereas potassium dihydrogen phosphate dihydrate (Sigma-Aldrich, Steinheim, Germany), orthophosphoric acid (Merck, Darmstadt, Germany) and acetonitrile (ACN) gradient grade (Sigma-Aldrich, Steinheim, Germany) were used. HPLC grade Acetonitrile and Methanol were procured from Merck Ltd.

A Shimadzu HPLC system consisting of the following components was utilized: pump LC – 20AD, vacuum degasser unit DGU – 20 A₅ and a UV/VIS detector SPD – 20 A. Separation was carried out on a LiChrosorb C 18 column (150 × 4 mm, particle size 5 µm) under reversed phase partition chromatographic conditions. The equipment was controlled by a PC using chromatographic software. The mobile phase was a 50:50% v/v, Acetonitrile: Phosphate buffer (0.136 g potassium dihydrogen phosphate in 1000 ml water, adjusted with ortho-phosphoric acid, pH = 3.0±0.1). The mobile phase was filtered through a 0.45 µm membrane filter and degassed by using a sonicator for about 10 min before use. The sample solutions were also filtered using 0.45 µm membrane filters. The mobile phase was delivered isocratically at a flow rate of 1.0 ml/min. The column was maintained at a temperature of 30 °C. The injection volume was 20 µl and the total run time was 20 minutes. The detection was carried out at 225 nm.

Accurately weighed quantity of 10 mg 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid (Compound 1a) was transferred in 100 ml volumetric flask, dissolved with a mixture of water and methanol (50:50) to volume, and mix. The concentration was 100 μg/ml. From the stock solution by further dilutions were prepared standard solutions within the concentration range 6.25 – 50.00 μg/ml.

Compound 1a (2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid) was accurately weighed and transferred into a 100 ml volumetric flask. Approximately 30 ml of a mixture of water and methanol (50:50) was added and the mixture was sonicated for 1 minute. Then the mixture was diluted to volume with a mixture of water and methanol (50:50). From the stock solution by further dilutions with buffer solutions (pH = 7.4 and pH = 9.00) were prepared the required working solutions. The solutions were thermostated at 37 °C for a total time 1440 min.

Solutions of the parent compounds (25 μg/ml) were also prepared in mixture of water and methanol (50:50).

27.22 g of potassium dihydrogen phosphate dihydrate was dissolved in 1000 ml of deionized water. 50.00 ml of the solution was mixed with 39.10 ml sodium hydroxide (0.2 mol/l) and 100 ml deionized water in a suitable container. The solution was adjusted to pH 7.4 (±0.02) and the final volume made up to 200.0 ml. 50.00 ml of the solution was mixed with 20.80 ml sodium hydroxide (0.2 mol/l) and 100 ml deionized water in a suitable container. The solution was adjusted to pH 9.0 (±0.02) and the final volume made up to 200.0 ml.

The structure of the analyzed molecule is presented on Fig. 1.

Figure 1. 

Structure of the analyzed compound (1a).

The synthesis of the analyzed structure is described elsewhere (Vladimirova et al. 2014) and presented on Fig. 2:

Figure 2. 

Synthesis of the analyzed 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid (5) (compound 1a).

During the chemical synthesis, it was observed an appearance of an impurity, eluting together with the final product. In an attempt to identify the possible mechanism by which this impurity is formed, the conditions, under which this impurity would appear and to follow the stability of the obtained 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid we optimized a high-performance liquid chromatography method.

The method was applied to separate and selectively detect and identify all process-related impurities and degradation products under different pH conditions at a temperature of 37 °C.

For following the degradation and appearance of impurities of previously synthesized 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid a simple, fast and selective RP-HPLC method was developed and applied. The presented method was validated in terms of precision, linearity, accuracy, selectivity, limits of detection, and quantification.

The method was validated according to ICH guidelines (ICH Guidelines Q2 R1 2005). The system suitability (i.e., repeatability of retention times and areas, number of theoretical plates, and resolution, Table 1), precision, linearity, accuracy and selectivity were evaluated during method validation. The parameters accuracy, precision, and selectivity were performed and evaluated for the analyzed product.

Limit of detection and quantification

The detection limit (LOD) is the lowest amount of analyte in the sample, which can be detected but not necessarily quantified as an exact value. The quantification limit (LOQ) is the lowest amount of analyte in the sample, which can be quantitatively determined with suitable precision and accuracy. The LOD and LOQ are calculated as given in Table 2.

The selectivity studies were performed against solvents used. The placebo solution was injected twice according to the parameters stated under the developed method. It was found that there was no interference between the analyte and placebo solutions (Fig. 3a, b).

Figure 3. 

a. Placebo chromatogram b. chromatogram of compound 1a.

Table 2 presents the equation of the regression line, correlation coefficient (r²) values of the slope and intercept between the peak areas and concentrations of 6.25–50.0 μg/ml with r² = 0.999 (Fig. 4). The calibration curve equation shows a good linearity curve which means that the linearity test is validated.

Figure 4. 

Linearity of Compound 1a.

For determination of the precision, the standard solution (25 µg/ml) was consecutively injected 6 times according to the above method. Assay % and RSD % values obtained are within the range of 98 – 102% (±2). The data given in Table 3 show a precise and valid method of analysis.

The accuracy test was applied at three different levels of concentrations with triple injecting for each sample (Table 4), the % of recovery equation is:

% Accuracy = [(recovered amount / actual amount) X 100]

The tested 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid was dissolved in water and methanol (50:50) and thermostated for a period of 1440 min at 37 °C. No new peaks appeared on the obtained chromatogram. Thus we considered that the product is completely purified from all possible process impurities.

An important factor influencing the performance of the molecules in the organism is their hydrolytic stability at physiological conditions, such as: a body temperature of 37 °C and physiological pH of 7.4 (blood plasma) and 9.0 (intestine) (Kong and Singh 2008).

The fully substituted pyrrole, as well as the connected with it methyl groups, are stable at moderate temperatures and in a wide range of pH values. The stability of the obtained structure will be determined by the stability of the identified as potential “vulnerable” group, positioned on 3^rd place in the pyrrole cycle ester group (Jordanov et al. 1994).

In order to determine the chemical stability and the stability of the ester group, positioned on 3^rd place at different pH of 7.4 (blood plasma) and 9.0 (intestine) and temperature of 37°C the analyzed structure was thermostated and stirred in a micro reactor at 37 °C for a total time of 1440 min. Aliquot samples of 0.5 ml of the analyzed solutions were taken at definite time intervals (15, 30, 60, 120, 240, 480 and 1440 min) and diluted to 1.5 ml with the corresponding mobile phase in a way that the concentration is in the range of 0.1 mg/ml and a 20 μl sample was injected. The corresponding chromatograms were obtained.

A 20 mg sample of the tested compound was weighed and dissolved in buffer pH 7.4. The obtained solution was stirred in a micro reactor at 37 °C for a total time of 1440 min. At the determined above time intervals, a 20 μl sample was drawn and injected into the apparatus. The obtained chromatogram is presented on Fig. 5.

Figure 6. 

Chromatogram of the analyzed compound 5 at 0 min (A) and at 30 min (B).

The performed analysis showed stability of the tested N-pyrrolylcarbocilyc acid and no impurities were detected under the discussed conditions. Thus 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid may be considered stable at this media.

A 20 mg sample of the tested compound was weighed and dissolved in buffer pH 9.0. The obtained solution was stirred in a micro reactor at 37°C for a total time of 1440 min. At the determined above time intervals a 20 μl sample was drawn and injected into the apparatus. Representative chromatograms, showing the behavior of the analyzed structure at 0 min and 30 min of the incubation are presented on Fig. 6.

Figure 5. 

Chromatogram of the analyzed compound 5.

The performed analysis showed the appearance of a new peak with retention time (t_R) of 7.33 min at 0 min of the analysis. The new substance stayed stable through the whole time of analysis (1440 min.), keeping the peak area constant.

For identification of this substance, the initial products, used in the synthesis were analyzed by the developed RP-HPLC method. The analysis showed, that the obtained during the incubation peak with t_R of 7.33 min, corresponds with the initial ethyl 2-acetyl-4-(4-chlorophenyl)-4-oxobutanoate (Fig. 7).

Figure 7. 

Chromatogram of the initial substance 3.

This lead to the conclusion, that at the low alkali media of pH 9.0 and temperature of 37°C the analyzed N-pyrrolylcarbocilyc acid degrades with cleavage of the pyrrole cycle and release of the initial ethyl 2-acetyl-4-(4-chlorophenyl)-4-oxobutanoate, identified as a process impurity.

On the other hand, the expected hydrolysis of the ester group at 3^rd position of the N-pyrrolylcarbocilyc acid did not occur. Thus this group may be considered stable under the applied conditions.