The model compounds were synthesized by Paal-Knorr condensation and characterized as described previously (Tzankova et. al. 2019a; Tzankova et. al. 2019b) All the chemicals and reagents used as starting materials were purchased from Merck (Darmstadt, Germany).

The necessary products for preparation of the mobile phase and buffers were obtained from Sigma-Aldrich (Steinheim, Germany).

The chromatographic system UltiMateDionex 3000 SD, Chromeleon 7.2 SR3 Systems, Thermo Fisher Scientific Inc was applied. The analysis was achieved on an analytical column 250 mm x 4.6 mm (Purospher^® STAR, RP-18, 5 μm) protected with a guard column (Hibar^®, RT 125-4). The column oven was conditioned at 25°C. The mixture of an acetonitrile, phosphate buffer pH 4.0 and methanol in a ratio 60:30:10 (v/v/v) was used as a mobile phase. The flow rate was 1.0 ml/min and the detector was set up to 272 nm. The injection volume was 20 μl with analysis time of 15 minutes.

Measured amount of 5.04 g of sodium hydrogen phosphate dihydrate and 3.01 g of potassium dihydrogen phosphate dihydrate were dissolved in 1000 ml of ultrapure water. Glacial acetic acid was used to adjust the pH to 4.0. The mobile phase buffer was filtered through a membrane filter (0.20 μm) using a Millipore glassfilter holder. The mobile phase buffer was used immediately after preparation or stored in the refrigerator in closed borosilicate glassbottles for a maximum of 24 hours.

Measured amount of 6.57 g of KCl (dissolved in water free from CO₂) and 119.0 ml of hydrochloric acid (0.1 mol/l) were dissolved in 1000 ml distilled water.

Measured amount of 2.38 g of sodium hydrogen phosphate dihydrate, 0.19 g of potassium dihydrogen phosphate dihydrate and 8.0 g NaCl were dissolved in 1000 ml distilled water.

Solution A – Measured amount of 6.18 g boric acid was dissolvedin 0.1 mol/l KCl. The obtained mixture was diluted up to 1000 ml with the same solvent.

Solution B – 0.1 mol/l NaOH.

Buffer pH 9.0 was prepared, where 1000 ml ofsolution. A are mixed with 420.0 ml of solution B.

A standard buffer with pH 13.0 (КCl/ NaOH) was used.

Measured amount of 0.0010 g sample of the analyzed compound was weighed and dissolved in 5 ml acetonitrile. The obtained mixture was diluted up to 50 ml with the corresponding buffers of pH 2.0, pH 7.4, pH 9.0 and pH 13.0. The obtained solutions were stirred at 37°C for a total time of 240 min. An aliquot 20 μl samples were drawn and injected into the HPLC system at the defined time intervals.

Mobile phase, consisting of CH₃CN/phosphate buffer pH 4.0/ CH₃OH = 60/30/10 (v/v/v) was prepared and applied.

In order to achieve good separation and proper chromatographic conditions for stability assessment, we have endeavored to obtain chromatographic conditions which would allow the determination of as many peaks as possible from the set of test samples. The most common separation conditions include solvent type, mobile phase composition and pH of the medium, column type and column temperature.The experimental conditions for stability analysis were achieved by planned / systematic study of parameters including the pH of the medium, components included in the mobile phase and ratios, gradient, flow rate, temperature, sample volume, injection volume and solvent type.

The developed RP-HPLC method was tested with respect to following validation parameters: specificity, precision, linearity, accuracy and selectivity.

Specificity of the chromatographic analytical procedure is the ability to measure the analyte response in the presence of all potential sample components such as the starting materials, intermediates in the synthesis, and inactive ingredients in the formulated products, and the degradation products. The selectivity of the method was evaluated by comparing the solution of the model compound (D_5d) with the solution of the corresponding hydrazide, aldehyde and used mobile phase alone and in mixture.

Precision is expressed as the standard deviation of the analytical results when the analysis is carried out in a laboratory by an operator using equipment over a relatively short time span. For precision of the method six independent sample solutions from evaluated hydrazone were used. Each sample was tested in triplicate. The final results are reported as relative standard deviations (RSD %).

The linearity of an analytical method can be defined as the ability of the method to obtain test results that are directly proportional to the analyte concentration, within a given range. The linearity of the method was determined in a concentration range of 5 - 40 μg/mL for hydrazone D_5d. An eight points covering the eight different concentrations of the analyzed compound over the evaluated concentration range were used to create the calibration curve. For processing the calibration data was used linear regression.

Accuracy is the closeness of the analytical results obtained by the analyses to the true values, and usually presented as a percent of nominal. The analyzed solutions were prepared using a placebo and stock solution of the tested structure. Accuracy is reported as a parameter recovery with relative standard deviations calculated from the analysis of six solutions prepared from the compound. Each solution was tested in triplicate.

The aim of this study is to develop, validate and apply a simple RP-HPLC method for stability determination of pyrrole-containing derivatives.

No significant interfering peaks (peak area >0.1%) was observed at the retention time of the analyzed hydrazone and mixtures in blank solution. In addition, no evidence of co-elution was noted using peak purity analysis for the tested compounds and mixtures.

The linearity of the method was observed in the concentration range of 5 μg/mL to 40 μg/mL for D_5d demonstrating its suitability for analysis. The goodness of fit evaluated through the correlation coefficient r² was found to be 0.9980, indicating a linear relationship between the concentration of analyte and area under the peak, as shown on Figure 6.

Figure 6. 

Linearity of the developed RP-HPLC D_5d stability indicating method.

The accuracy of an analytical procedure expresses the closeness of results obtained by that method to the true value. The results of accuracy testing showed that the method is accurate within the acceptable limits. The percentage recovery and RSD were calculated for all active ingredients; all the results are within limits. Acceptable accuracy was within the range of 99.98% to 100.4% recovery and not more than 2.0% RSD, as demonstrated in Table 1.

Table 1.Download as CSV 

Evaluation of the accuracy of the method developed in this study.

Compound	Spiked level (μg/mL)	Replicate number	Recovery (μg/mL)	% mean recovery	% RSD
D_5d	5	1	5.05	100.4	0.72
		2	4.98
		3	5.03
	20	1	19.99	100.2	0.16
		2	20.04
		3	19.98
	40	1	40.07	99.98	0.23
		2	39.89
		3	40.02

Precision of an analytical method is defined as “the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions,” and it is normally expressed as the relative standard deviation. The results of repeatability and intermediate-precision testing showed that the method is precise within the acceptable limits.The RSD was calculated and was found that the results are within limits. Precision was not more than 2.0% RSD, as demonstrated in Table 2.

Table 2.Download as CSV 

Evaluation of precision of the method developed in this study.

Intermediate precision (ruggedness)
Replicate number	Recovery (μg/mL)
1	19.99
2	20.01
3	20.03
4	19.98
5	20.04
6	19.87
Mean recovery	19.99
% RSD	0.31

The limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated, while the limit of quantification (LOQ) is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision.The method showed a LOD of 0.20 μg/mL and a LOQ of 0.40 μg/mL.

An important factor influencing the performance of the molecules in the organism is their hydrolytic stability at physiological conditions, such as: body temperature of 37 °C and physiological pH of 2.0 (in stomach), 7.4 (in blood plasma) and 9.0 (in intestine) (Georgieva et al. 2012)

The selected model compound was subjected to analysis following the requirements for physiological stability evaluation. The tested D_5d was dissolved in the corresponding buffers, mimicking physiological conditions and stirred at 37°C for 240 min.

For both types of stability evaluation four solutions were prepared according to the above mentioned recipe. The obtained chromatograms for the tested D_5d for the physiological conditions were recorded and are presented on Figure 7 and Figure 8 respectively.

In the performed investigations an appearance of new peaks, corresponding to the retention times of the initial hydrazide and the corresponding aldehyde are observed in the chromatogram of D_5d in acidic (pH 2.0) (Figure 7) media. This led us to conclude that this substance is susceptible to hydrolysis at these conditions and as a result are formed the initial reagents.

Figure 7. 

Chromatograms indicating the behavior of D_5d in the presence of buffer with pH 2.0 and at 37°C at 0^th min (A) and at 30^th min (B).

No additional peaks appeared in the chromatograms of the analysis performed in the neutral buffer of pH = 7.4 and alkali buffer of pH 9.0 (Figure 8) and temperature of 37°C for the evaluated period of time, which led us to conclude that the structure is stable at these conditions.(33–35).

Figure 8. 

Chromatograms indicating the behavior of D_5d in the presence of buffer with pH 9.0 and at 37°C at 0^th min (A) and at 210^th min (B).

In addition the chemical stability was also evaluated in the same manner by following the same procedure, using buffer of pH = 13.0. The corresponding chromatogram is presented on Figure 9.

Figure 9. 

Chromatograms indicating the behavior of D_5d in the presence of buffer with pH 13.0 and at 37°C at 0^th min (A) and at 30^th min (B).

The chromatogram shows appearance of new peaks at the retention times of the initial hydrazide and the corresponding aldehyde. This shows that the structure is liable to hydrolysis at strong alkali media. The result is cleavage of the hydrolytically liable hydrazone group, as suggested previously.