All of the solvents used in the current work were purchased from Sigma Aldrich and are with European pharmacopoeia purity. Potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate and orthophosphoric acid were obtained from Sigma-Aldrich and are of analytical grade. The evaluated hydrazide-hydrazone derivative was synthesized as described in a recent work by Tzankova et al. (Tzankova et al. 2020)

A male Wistar rats with body weights around 200 g were used. The latter were housed in plexiglass cages at room temperature in 12/12 light/dark cycle. The animals were purchased from the National Breeding Centre, Sofia, Bulgaria and all performed procedures were approved by the Institutional Animal Care Committee with accordance with European Union Guidelines for animal experimentation.

Sodium pentobarbital (0.2 mL/100 g) was employed for the anesthesia of the rats. A modified method described by Fau et al. (Fau et al. 1992) was used for in situ liver perfusion and cell isolation. After portal catheterization, the liver was perfused with 100 mL HEPES buffer (pH = 7.85), containing 10 mM HEPES, 142 mM NaCl, 7 mM KCl, 5 mM glucose and 0.6 mM EDTA (pH = 7.85), followed by 200 ml HEPES buffer (pH = 7.85), with a final addition of 200 ml HEPES buffer containing collagenase type IV (50 mg/200 mL) and 7 mM CaCl₂ (pH = 7.85).

The liver was excised, minced into small pieces and hepatocytes were dispersed in 60 mL Krebs-Ringer-bicarbonate (KRB) buffer (pH = 7.35), containing 1.2 mM KH₂PO₄, 1 mM CaCl₂, 1.2 mM MgSO₄, 5 mMKCl 5 mM NaHCO₃, 4.5 mM glucose and 1% bovine serum albumin. After the initial filtration, the hepatocytes were centrifuged at 500 g for 1 minute and washed three times with KRB buffer. Cells were counted under the microscope and the viability was assessed by Trypan blue exclusion (0.05%), which was calculated to be 89% in average (Fau et al. 1992). Cells were diluted with a KRB, to make a suspension of about 3 × 10⁶ hepatocytes/mL. Incubations were carried out in 25 mL Erlenmeyer flasks with each containing 3 mL of the cell suspension (i.e. 9 × 10⁶ hepatocytes). The latter procedures were performed in a 5% CO₂ + 95% O₂ atmosphere.

The hepatocytes were incubated at KRB buffer with 50 µM hydrazide- hydrazone compound for 2 h. Subsequently, the obtained cell emulsion was precipitated with 1 mL HPLC grade methanol and centrifugated for 15 min at 14 000 rpm. The supernatant was double filtered through PVDF sterile syringe filters (through 0.47 µm, and through 0.22 µm).

The applied chromatography system was constructed out of UltiMateDionex 3000 SD pump connected to UltiMateDionex DAD 3000 detector. Separation of compounds was performed on a 15 × 0.46 cm, 5 μm particle size, Purospher C18 Column protected by a C18 guard column (SecurityGuard HPLC Cartridge System; Phenomenex). The former was conditioned at 25 °C in a column oven. Data were recorded and evaluated by Chromeleon 7 software.

The mobile phase consisted of acetonitrile: buffer pH 3.5: methanol in ratio 57/38/5 (v/v/v). The mobile phase buffer was prepared according to the European Pharmacopea and filtered through a membrane filter (0.20 μm) using a Millipore glass filter holder. The flow rate was set to 0.8 mL/min with injection volume of 10 µL.

The specificity is defined as the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. The specificity of the developed technique was confirmed by checking of test solutions and the special solvent chromatograms (ICH Guidlines Q2 R1 2005).

The linearity of the analytical method is based on the direct proportional dependence of the analytical signal on the concentration (amount) of the analyte in the sample within the analytical area of the method (ICH Q2 R1 2005). In order to prove the linearity of a method, the correlation coefficient must be above 0.99.

To assess the system suitability, retention time of six injections of the standard solution, tailing factor and theoretical plates were used.

Limit of Detection (LOD) is the smallest amount of analyte in a sample which can be identified, however not necessarily accurately quantified. Limit of Quantification (LOQ) is the minimum amount of a substance in a sample that can be quantified with the appropriate precision and accuracy. Limit of Detection (LOD) and Limit of Quantitation (LOQ) were resoluted using calibration curve method by applying the following equals: LOD = 3.3 × Sa/b and LOQ = 10 × Sa/b (ICH Q2 R1 2005).

The ICH Q2 R1 Guideline defines the precision procedure as the closeness of results between a series of measurements for a sample taken from the same homogeneous solution under the specified conditions.

The accuracy of an analytical procedure expresses the closeness of the results obtained by the method to the true value (Borman and Elder 2017). To evaluate the accuracy of the proposed method, percentage recovery and %RSD values should be ranging from 98.0% to 102.0%, and not more than 2.0%, respectively.

The stability of the targeted pyrrole hydrazone was evaluated through an RP-HPLC method developed and applied by Tzankova et al. (Tzankova et al. 2019b). This pointed our attention to employ in this study also a reversed phase HPLC method. In addition a column with geometric parameters 15 × 0.46 cm, 5 μm particle size, Purospher C18 Column with Phenomenex SecurityGuard C18 column was selected. The composition of the mobile phase was based on the analyte properties and blank solution content, using as a starting point the mobile phase described in Tzankova et.al. (Tzankova et al. 2019b), where for the hydrazone compound was assigned a peak with retention time of 6.110 min and for its hydrolysis product aldehyde – a retention time of 1.283 min. The method was found to be not applicable for our investigation, which required a reduction of the organic solvents - acetonitrile and methanol and increase of the volume of the pH 3.5 buffer. The performed changes determined as most suitable parameters for achieving good separation and suitable peak forms to be: mobile phase - CH₃CN/phosphate buffer pH 3.5/ CH₃OH: 57/38/5 (v/v/v). The flow rate, the temperature and the wavelength were also altered. The flow rate of 1.0 ml/min was found to be most appropriate. A temperature of 25 °C was found to be suitable for the good separation. The preliminary UV/VIS method developed by us identified 272 nm as most adequate wavelength.

Specificity of the method was evaluated by injecting 20 μl solutions of standard, sample, and blank separately. The blank solution does not give interfering peaks at the retention time of analyzing compound.

Linearity was studied by analysis of standard solutions of the hydrazide- hydrazone compound at different concentrations using methanol as a solvent. The obtained data was analyzed after conducting 5 different levels of dilution (35, 70, 105, 140, 175 μg/mL). Thereafter, the results were used to build a calibration curve, with a subsequent calculation of the linear equation and the correlation coefficient. The aforementioned parameters are given in Table 1.

The obtained results confirm the linearity of the constructed method.

System suitability data was evaluated based on the chromatogram of the hydrazone solution. Precision was assessed by analyzing six samples with concentration of 70 µg/mL and the Relative standard deviation was calculated. Results are summarized in Table 2.

The accuracy of the method was determined by recovery studies applying three concentration levels (50%, 100%, and 150%) and three samples from each concentration were injected. The obtained results demonstrated percentage recovery in the range of 99.9–100.8% at all three levels (Table 3). The percentage recovery and the %RSD values were within the accepted limits which affirmed the applicability of the method.

As most appropriate compound for the preliminary evaluation of the metabolic transformations in isolated rat hepatocyte suspension was selected (E)-ethyl 5-(4-bromophenyl)-1-(1-(2-(2-hydroxybenzylidene)hydrazinyl)-1-oxo–3–phenylpropan-2-yl)–2–methyl–1H-pyrrole-3-carboxylate (Figure 1).

Figure 1. 

Structure of the evaluated pyrrole hydazide-hydrazone.

The developed RP-HPLC method was applied for determination of the initial hydrazide and salicyl aldehyde, which were hydrolytical products and possible metabolites. The identified retention times of the tested compounds are 5.74 min for the N-pyrrolylhydrazide and 2.10 min for the aldehyde.

For determination of the possible hydrazone`s metabolites the developed and optimized RP-HPLC-DAD method was applied. In the performed investigation after 30 min incubation in isolated rat hepatocytes, three new peaks appeared (Figure 2A). Two of the detected peaks corresponded to the retention times of the identified in the previous analysis salicyl aldehyde (2.08 min) and initial N-pyrrolylhydrazide (5.75 min) (Figure 2A).

Figure 2. 

Chromatograms demonstrating the hepatocytic metabolism of analyte at 30^th min (A) and at 60^th min (B).

As demonstrated in Figure 2 B, the concertation of studied molecule dropped to 38 μg while the area of the other peaks was increased in the 60^th minute mark. After 2 hours of incubation, аn appearance of new peak with retention time of 9.14 was detected (Figure 3).

Figure 3. 

Chromatogram demonstrating the hepatocytic metabolism of analyte at 120^th min.

The retention time of the new peak is not ascribable to any of the known and used as references molecules. This leads to the conclusion of possible formation of new unidentified metabolic product.

In addition it was of interest to try to predict the possible structure of the unidentified metabolic derivative. For this purpose a virtual preliminary metabolic evaluation with the online server SMARTCyp (Rydberg et al. 2010) was done. The results identified 14 possible metabolites – 11 hydroxylated molecules and 3 epoxide derivates as presented in Table 4.

Table 4.

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Metabolism prediction of the title compound.

Substitions	Reaction	Enzymes
R1-11 – OH	hydroxylation	Cyp3A4
R1-3 – O	epoxidation	Cyp3A

The performed RP-HPLC evaluation and in silico prediction determined that the metabolic transformation of the evaluated (E)-ethyl 5-(4-bromophenyl)-1-(1-(2-(2-hydroxybenzylidene) hydrazinyl)- 1 oxo–3–phenylpropan-2-yl) –2–methyl–1H-pyrrole-3-carboxylate is associated with hydrolytic degradation of the newly formed hydrazide-hydrazone group and additional hydroxylation or epoxidation in the presence of hepatocyte cells. The identification of the newly detected peak at 9.14 min will be a subject of additional research.