All reagents for chromatographic procedure and sample preparation were purchased from Merck Darmstadt, Germany. The methanol and acetonitrile used in analyses were HPLC gradient grade and were obtained from Fisher Chemical, UK. The phosphate buffer was prepared by dissolving 5.04 g Na₂HPO₄ and 3.01 g KH₂PO₄ in 1 L distilled water. To adjust the pH to 3.5, the orthophosphoric acid was used. The selected molecules 11b and 12b used in this study were previously synthesized and described in works of Bijev and Georgieva (2010) and Georgieva et al. (2012).

Synaptosomes were prepared from the brains of adult male Wistar rats through multiple, subcellular fractionation using a Percoll gradient, as previously described in work from Taupin et al. 2014 Thereafter, the isolated subcellular fraction was incubated for 2 hours in an appropriate buffer with 200 µL of 1 mM solutions of selected compounds. The procedure for sample purification included a rapid and simple protein precipitation using HPLC grade methanol followed by centrifugation at 14.000 rpm and filtration. The samples were initially analyzed using UltiMateDionex 3000 equipped with a diode array detector set up at 279 nm. The data was processed by Chromeleon 7.2 software. The analytes were separated on a Purospher STAR RP-18 (4.6 × 12.5 cm, particles size 5 µm) column with 0.8 mL/min flow rate of the mobile phase, which consisted of acetonitrile: phosphate buffer pH 3.5: methanol in ratio 42/36/22 (v/v/v).

Method validation was carried out according to the requirements of the ICH guideline. The parameter linearity was evaluated using a calculated correlation coefficient, which value must be greater than 0.99. To estimate the repeatability of the method, relative standard deviation (RSD≤2) was generated from peak areas of 6 samples at 100% working concentration. The validation proceeded with an assessment test for accuracy. This necessitated triplicate analysis of solutions at three concentration levels (50, 100 and 150%) with subsequent determination of the Recovery %. The limits of detection (LOD) ant quantitation (LOQ) were calculated based on the method of the standard deviation of a response and the slope using the following equations:

$LOQ=\frac{10{\alpha }_b}{S}$ $LOD=\frac{3{\alpha }_b}{S}$

where σ_b- the standard deviation of the response; S- the slope of the calibration curve

Our previous study (Mateeva et al. 2022) involved development and application of RP-HPLC method for possible hepatic metabolism evaluation of the pyrrole-containing hydrazide-hydrazone with similar structures (Tzankova et al. 2020). The separation was performed on a column Purospher C18 (15 × 0.46 cm, 5 μm) conditioned at 25 °C. The elution was isocratic with a mobile phase of acetonitrile: buffer pH 3.5: methanol in ratio 57/38/5 (v/v/v). This pointed our attention to utilize a shorter column (12.5 cm) of Purospher, Phenomenex with the same mobile phase. The results demonstrated retention times of about 5 minutes for both hydrazones. Due to more polar properties of the expected metabolites, we concluded that the method was not suitable for our investigation. Thus, the experiments continued with a change in the volume ratios of the mobile phase. Several diverse combinations were applied in order to achieve the optimal retention times and good peak’s shape of the 11b and 12b. This was accomplished through a mobile phase, which consisted of acetonitrile: buffer pH 3.5: methanol in ratio 42/36/22 (v/v/v). The temperature in the column compartment was also switched to 20 °C. The recording of UV/VIS spectra of the selected molecules revealed the most adequate wavelength at 279 nm.

The above mentioned changes in chromatographic parameters let to further validation of the method.

Validation was prepared according to ICH guideline Q2 (R2) and the parameters as specificity, linearity, range, accuracy, precision and sensitivity were established.

The specificity of the method was evaluated through analysis of the blank solution containing isolated synaptosomes. The obtained chromatogram (Fig. 2) of the purified and pre-treated biological sample did not reveal any interfering peaks ascribable to the protein contents.

Figure 2. 

Chromatogram of the pre-treated biological media.

The lack of peaks with retention times close to, or equal to the ones of the analyzed samples determined the procedure as suitable for subsequent investigation.

To estimate the validation parameters linearity and range, six working solutions of each hydrazone with linearly increasing concentration were analyzed. The results were used to generate the calibration curves by plotting the peak area against the concentrations of 11b and 12b. Subsequently, the linear equation and correlation coefficient were calculated. The results are summarized in Table 1 and demonstrated good correlation in the investigated range.

Sensitivity was also evaluated by calculating the Limit of Detection (LOD) and Limit of Quantification (LOQ) using the aforementioned equations. The values of the latter parameters are 0.8825 and 2.6743 µM for 11b and 1.1022 and 3.0402 µM for 12b, respectively.

Precision of the method was estimated via calculation of the RSD. The obtained results of the latter, with values 0.5326 for 11b and 0.4952 for 12b, proved acceptable repeatability of the method. Accuracy was established by triplicate analyzing three different levels (50,100 and 150%) for both hydrazones. The results are presented in Table 2.

In an attempt to identify the possible appearance of biotransformation and the kinetics of the process, the selected molecules were incubated for 2 hours in isolated rat brain synaptosomes and aliquot samples were collected every 30 min. The drawn quantities were subjected to the purification process indicated in the Materials and Methods section. Prior to analysis, the supernatant was double filtered through PVDF sterile syringe filters (through 0.47 μm, and through 0.22 μm) and a volume of 10 µL was injected into the chromatographic system.

The obtained chromatograms (Figs 3, 4) disclosed negligible decrease in concentration of the analytes and absence of new peaks.

Figure 3. 

Chromatograms of compound 11b at 0 (A) and 120^th (B) minute.

Figure 4. 

Chromatograms of compound 12b at 0 (A) and 120^th (B) minute.

The observed chromatographic behavior of the tested hydrazones is believed to be due to the lack of a target group for the monoamine oxidase - the main enzyme in isolated subcellular culture.