In this study, the effects of the parameters, ethanol/methanol concentration, mobile-phase pH, and column temperature, on the retention behavior of metronidazole and albendazole were investigated using a miniaturized green reverse phase liquid chromatography (green RPLC) method based on a central composite design (CCD). The multiple effects of these parameters on the retention behavior of the studied compounds were evaluated to obtain optimal liquid chromatographic separation conditions for their simultaneous determination. In contrast, the individual effects of these parameters on the studied compounds were evaluated to obtain some physicochemical constants (dissociation and thermodynamic constants). The optimum chromatographic conditions were found employing CCD and Derringer’s desirability function. Optimum analysis conditions were determined as an ethanol–water mixture containing 9.5% (v/v) ethanol with pH adjusted to 4.5 and a methanol–water mixture containing 10% methanol with pH adjusted to 4.5. The Supelco Ascentis RP Amide (150 × 4.6 mm, 2.7 µm I.D.) column was preferred for the determination of the investigated compounds in both hydro-organic mixtures. The column temperature was kept constant at 37 °C. The method developed for the simultaneous determination of the investigated compounds demonstrated a correlation coefficient of 0.999 and linearity in the concentration range of 0.5–3.0 µg/mL, with acceptable sensitivity and accuracy values in the ethanol–water hydro-organic mixture, resulting in shorter separation times. Validated according to the ICH Q2 (R1) guideline, the method was successfully applied to the tablet dosage preparation of metronidazole. The dissociation constant values (pKa) and thermodynamic parameters (<InlineEquation ID="IEq1"> <InlineMediaObject> <ImageObject Color="BlackWhite" FileRef="10337_2025_4442_Article_IEq1.gif" Format="GIF" Height="14" Rendition="HTML" Resolution="72" Type="Linedraw" Width="37" /> </InlineMediaObject> <EquationSource Format="TEX">$\Delta {H}^{o}$</EquationSource> <EquationSource Format="MATHML"><math> <mrow> <mi mathvariant="normal">Δ</mi> <msup> <mrow> <mi>H</mi> </mrow> <mi>o</mi> </msup> </mrow> </math></EquationSource> </InlineEquation>, <InlineEquation ID="IEq2"> <InlineMediaObject> <ImageObject Color="BlackWhite" FileRef="10337_2025_4442_Article_IEq2.gif" Format="GIF" Height="15" Rendition="HTML" Resolution="72" Type="Linedraw" Width="33" /> </InlineMediaObject> <EquationSource Format="TEX">${\Delta S}^{o}$</EquationSource> <EquationSource Format="MATHML"><math> <msup> <mrow> <mi mathvariant="normal">Δ</mi> <mi>S</mi> </mrow> <mi>o</mi> </msup> </math></EquationSource> </InlineEquation> and <InlineEquation ID="IEq3"> <InlineMediaObject> <ImageObject Color="BlackWhite" FileRef="10337_2025_4442_Article_IEq3.gif" Format="GIF" Height="19" Rendition="HTML" Resolution="72" Type="Linedraw" Width="41" /> </InlineMediaObject> <EquationSource Format="TEX">${\Delta G}^{o})$</EquationSource> <EquationSource Format="MATHML"><math> <mrow> <msup> <mrow> <mi mathvariant="normal">Δ</mi> <mi>G</mi> </mrow> <mi>o</mi> </msup> <mrow> <mo stretchy="false">)</mo> </mrow> </mrow> </math></EquationSource> </InlineEquation> of the compounds were estimated using the equation obtained from CCD within the limits of the selected parameters. The data obtained from this study agrees with pKa values obtained from other analytical methods using more toxic solvents.

Application of a Miniaturized Central Composite Design-Based Green RPLC Method in Evaluating the Retention Behavior of Metronidazole and Albendazole: The Optimization and Physicochemical Constants

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发表日期：

2025年9月19日

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Ali Onur Boyalıoğlu,
Y. Doğan Daldal,
Ebru Çubuk Demiralay

摘要

In this study, the effects of the parameters, ethanol/methanol concentration, mobile-phase pH, and column temperature, on the retention behavior of metronidazole and albendazole were investigated using a miniaturized green reverse phase liquid chromatography (green RPLC) method based on a central composite design (CCD). The multiple effects of these parameters on the retention behavior of the studied compounds were evaluated to obtain optimal liquid chromatographic separation conditions for their simultaneous determination. In contrast, the individual effects of these parameters on the studied compounds were evaluated to obtain some physicochemical constants (dissociation and thermodynamic constants). The optimum chromatographic conditions were found employing CCD and Derringer’s desirability function. Optimum analysis conditions were determined as an ethanol–water mixture containing 9.5% (v/v) ethanol with pH adjusted to 4.5 and a methanol–water mixture containing 10% methanol with pH adjusted to 4.5. The Supelco Ascentis RP Amide (150 × 4.6 mm, 2.7 µm I.D.) column was preferred for the determination of the investigated compounds in both hydro-organic mixtures. The column temperature was kept constant at 37 °C. The method developed for the simultaneous determination of the investigated compounds demonstrated a correlation coefficient of 0.999 and linearity in the concentration range of 0.5–3.0 µg/mL, with acceptable sensitivity and accuracy values in the ethanol–water hydro-organic mixture, resulting in shorter separation times. Validated according to the ICH Q2 (R1) guideline, the method was successfully applied to the tablet dosage preparation of metronidazole. The dissociation constant values (pK_a) and thermodynamic parameters ( $\Delta {H}^{o}$ $Δ H^{o}$ , ${\Delta S}^{o}$ ${Δ S}^{o}$ and ${\Delta G}^{o})$ ${Δ G}^{o})$ of the compounds were estimated using the equation obtained from CCD within the limits of the selected parameters. The data obtained from this study agrees with pK_a values obtained from other analytical methods using more toxic solvents.