The necessary products for preparation of the mobile phase and solutions are of analytical grade, obtained from Sigma-Aldrich, Steinheim, Germany.

The chromatographic separation was performed on a modular HPLC system UltiMate Dionex 3000 SD, Chromeleon 7.2 SR3 Systems, Thermo Fisher Scientific Inc. The separation was achieved isocratically with chiral ACE®Equivalence™ С18 (5 μm), 250 mm x 4.6 mm, 5 μm column eluted with a mixture of methanol, water and 0.5 M potassium dihydrogen phosphate buffer pH 7.4, 50:46:4 v/v/v, as the mobile phase at flow rate of 1.0 ml/min. The mobile phase was filtered through a 0.45 μm membrane filter and degassed. Detection was carried out by absorbance at 272 nm. The analysis was carried out at column temperature of 25^oC and injection volume of 20 μl.

CH₃OH:H₂O:phosphate buffer pH 7.4 = 50:46:4 (v/v/v).

The used buffer was prepared according to the methodology described in European Pharmacopoea 7.0 (2016).

A 2 mg sample of the tested compound was weighed and dissolved in 10 ml of methanol. A 2.5 ml portion of this solution was diluted to 25.0 ml with methanol and 20 μl of the obtained solution was injected into the apparatus.

The main purpose of this work was to develop a fast Reverse Phase HPLC method to separate and quantify the two enantiomers in 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluorobenzylidene)propanehydrazide. During the method development, several chiral HPLC columns were selected on the basis of the chemical structure and location of the chiral centre of the analyte and also on the characteristics of the chiral phase of the analytical columns. The best chiral selectivity and resolution were obtained on an ACE®Equivalence™ С18 column. Therefore, the ACE®Equivalence™ С18 column was selected for further optimisation.

For optimisation of the mobile phases, the effect of a variety of organic modifiers such as methanol, ethanol, glacial acetic acid and phosphate buffers with different pH were investigated. The finally selected mobile phase was methanol : water : phosphate buffer pH 7.4 (50:46:4, v/v/v). Table 1 summarises the final chromatographic conditions. The final method was then used for analysis of both 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluorobenzylidene)propanehydrazide isomers in the racemic mixture.

Table 1.Download as CSV 

Final chromatographic conditions of the method.

Chiral HPLC column	ACE®Equivalence™ С18 (250 x 4.6 mm), 5µm
Mobile phase	CH3OH:H2O:phosphate buffer pH 7.4 = 50:46:4 (v/v/v)
Temperature	25 oC
Flow rate	1.0 ml/min
Wavelength	272 nm
Injection volume	20 μl
2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N'-(3-fluorobenzylidene)propanehydrazide	0.2 mg/ml

As indicated in Figure 2, the separation of the two enantiomers was obtained using isocratic elution by use of CH₃OH:H₂O:phosphate buffer pH 7.4 = 50:46:4 (v/v/v) as mobile phase and an ACE®Equivalence™ С18 column as the chiral stationary phase.

Figure 2. 

A typical chromatogram of 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluorobenzylidene)propanehydrazide racemate analysed using the final chromatographic conditions.

The method was validated according to ICH Q2 (R1) guidelines (ICH 2005). The system’s suitability, precision, linearity, accuracy and selectivity were evaluated during method validation.

The system’s suitability requirements (blank baseline: no interfering peaks above 0.1% level at the retention time of two 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluorobenzylidene)propanehydrazide enantiomers, 0.2% standard solution having signal-to-noise ratio (S/N) ≥ 10 for each enantiomer; standard solution preparation agreement (≤ 2.0%), resolution factor (≥ 1.2), injector agreement (difference between two injections ≤ 2.0%)) were met prior to performing the method validation experiments.

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

The limit of quantification (LOQ) and limit of detection (LOD) were calculated from the standard deviations and slopes of the responses, using the signal-to-noise ratio. The LOQ for 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluorobenzylidene)propanehydrazide was found to be 0.02 μg/ml, while the LOD was 0.002 μg/ml.

In this study, “Enantiomer 2” is treated as the main structure and “Enantiomer 1” is treated as impurity. The linearity of both enantiomers was evaluated. Since “Enantiomer 2” was treated as the main structure, its linearity was evaluated from 25% to 150% of the nominal concentration. Linear regression was used to process the calibration data. The correlation coefficients of linearity were 0.989 for 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluorobenzylidene)-propanehydrazide, which indicated good correlation between the peak areas and the range of concentrations studied (Figure 3).

Figure 3. 

Linearity of the developed chiral RP-HPLC method.

A linear least square analysis of the data gave a correlation coefficient (R²) of 0.989. The y-intercept obtained was ≤ 0.5% of the 100% level, indicating that there is no significant bias for quantification for “Enantiomer 2”. The method is considered accurate for “Enantiomer 2” as per ICH guidelines.

“Enantiomer 1” is treated as an impurity and the linearity and recovery were studied together by preparing a series of “Enantiomer 1” solutions. A linear least square analysis of the data gave a correlation coefficient (R²) of 0.988. The y-intercept obtained was ≤ 3.0% of the 100% level indicating that there is no significant bias for quantification for “Enantiomer 1”. The method is proven to be linear within the investigated range.

The method’s precision was evaluated by analysing six solutions of Enantiomer 2 at 100% level. The enantiomer purity value was calculated for each preparation. The average enantiomer purity was 99.3% and RSD = 0.645% (n = 6), thus meeting the criteria of RSD ≤ 2%. These results demonstrate that the method is precise for determination of the enantiomer purity of the 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluoro-benzylidene)propanehydrazide samples.

The developed chiral RP-HPLC method was applied for identification and separation of enantiomers in newly synthesised 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluoro-benzylidene)propanehydrazide. The result is presented in Figure 4.

Figure 4. 

Representative chromatogram of the analyzed 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purine-7(6H)-yl)-N’-(3-fluoro-benzylidene)propanehydrazide.

The chromatogram shows that the developed method is applied for successful separation and identification of the possible enantiomers of theophylline- and xanthine-based hydrazide-hydrazones.