Suggest Mobile phase for Impurity of Oteseconazole:SHR8008X:SHR8008JA retention time with good separation
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Composition isocrated / Gradient for
Oteseconazole:SHR8008X:SHR8008JA retention time with good separation.
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Based on the chemical properties of Oteseconazole (VT-1161)—which is a tetrazole-based antifungal agent containing pyridine and fluorinated phenyl rings—and typical separation strategies for its related impurities (SHR8008 series), here is a robust starting point for HPLC method development.
Since specific impurity codes like SHR8008X and SHR8008JA are often proprietary manufacturing intermediates or degradation products, a Gradient Method is strongly recommended over an isocratic one to ensure you capture both early-eluting (polar) and late-eluting (non-polar) impurities.
Recommended Gradient Method (Best for Impurity Profiling)
This method utilizes a C18 column with a standard acidic buffer to suppress ionization of the basic nitrogen atoms found in Oteseconazole, ensuring sharp peak shapes.
Chromatographic Conditions:
Column: C18 Column (e.g., Agilent Zorbax Eclipse Plus or Waters XBridge), 150 x 4.6 mm, 3.5 um or 5 um.
Flow Rate: 1.0 mL/min
Detection (UV): 210 nm (for detecting impurities with weak chromophores) or 260 nm (more specific to the drug).
Column Temperature: 30^\circ\text{C} - 40^\circ\text{C}
Mobile Phase Composition:
Mobile Phase A: 0.1% Orthophosphoric Acid (H_3PO_4) in Water OR 10mM Potassium Dihydrogen Phosphate (KH_2PO_4) adjusted to pH 3.0.
Mobile Phase B: Acetonitrile (ACN).
Time (min) Mobile Phase A (%) Mobile Phase B (%) Purpose
0.0 70 30 Initial hold for polar impurities
5.0 70 30 Isocratic hold
25.0 10 90 Linear ramp to elute Oteseconazole & hydrophobic impurities
30.0 10 90 Wash step
30.1 70 30 Return to initial
35.0 70 30 Re-equilibration
Alternative Isocratic Method (For Quick Assay)
If you require an isocratic method specifically (though less effective for separating complex impurities like SHR8008X/JA), try this composition.
Composition: Buffer : Acetonitrile (45 : 55 v/v)
Buffer: 0.1% Orthophosphoric acid or 10mM Ammonium Acetate (pH 4.5).
Note: If Oteseconazole elutes too quickly (near the void volume), decrease the Acetonitrile to 45% or 40%. If it takes too long, increase Acetonitrile to 60%.
Critical Considerations for Optimization
To achieve the "good separation" requested in the prompt, consider these factors if the initial run does not resolve SHR8008X from SHR8008JA:
1. pH Control is Key
Oteseconazole contains basic nitrogen atoms (tetrazole/pyridine moieties).
If peaks are tailing: Ensure your buffer pH is roughly 3.0. This keeps the basic nitrogens protonated and prevents them from interacting with unreacted silanols on the column stationary phase.
Buffer Choice: If you are using Mass Spectrometry (LC-MS), swap the Phosphoric Acid for 0.1% Formic Acid or Ammonium Formate.
2. Modifying Selectivity
If the impurities (SHR8008X and JA) co-elute with the main peak:
Change the Organic Modifier: Substitute Methanol for Acetonitrile. Methanol has different selectivity (protic solvent) compared to Acetonitrile (aprotic) and often resolves positional isomers better.
Change the Column Chemistry: If a standard C18 fails, try a Phenyl-Hexyl column. The pi-pi interactions from the phenyl column often help separate aromatic compounds like azole antifungals better than a standard alkyl chain.
3. Sample Diluent
Dissolve your sample in a mixture of Water:Acetonitrile (50:50). Avoid dissolving in 100% Acetonitrile if your starting gradient conditions are high aqueous, as this causes peak distortion (fronting).
Since specific impurity codes like SHR8008X and SHR8008JA are often proprietary manufacturing intermediates or degradation products, a Gradient Method is strongly recommended over an isocratic one to ensure you capture both early-eluting (polar) and late-eluting (non-polar) impurities.
Recommended Gradient Method (Best for Impurity Profiling)
This method utilizes a C18 column with a standard acidic buffer to suppress ionization of the basic nitrogen atoms found in Oteseconazole, ensuring sharp peak shapes.
Chromatographic Conditions:
Column: C18 Column (e.g., Agilent Zorbax Eclipse Plus or Waters XBridge), 150 x 4.6 mm, 3.5 um or 5 um.
Flow Rate: 1.0 mL/min
Detection (UV): 210 nm (for detecting impurities with weak chromophores) or 260 nm (more specific to the drug).
Column Temperature: 30^\circ\text{C} - 40^\circ\text{C}
Mobile Phase Composition:
Mobile Phase A: 0.1% Orthophosphoric Acid (H_3PO_4) in Water OR 10mM Potassium Dihydrogen Phosphate (KH_2PO_4) adjusted to pH 3.0.
Mobile Phase B: Acetonitrile (ACN).
Time (min) Mobile Phase A (%) Mobile Phase B (%) Purpose
0.0 70 30 Initial hold for polar impurities
5.0 70 30 Isocratic hold
25.0 10 90 Linear ramp to elute Oteseconazole & hydrophobic impurities
30.0 10 90 Wash step
30.1 70 30 Return to initial
35.0 70 30 Re-equilibration
Alternative Isocratic Method (For Quick Assay)
If you require an isocratic method specifically (though less effective for separating complex impurities like SHR8008X/JA), try this composition.
Composition: Buffer : Acetonitrile (45 : 55 v/v)
Buffer: 0.1% Orthophosphoric acid or 10mM Ammonium Acetate (pH 4.5).
Note: If Oteseconazole elutes too quickly (near the void volume), decrease the Acetonitrile to 45% or 40%. If it takes too long, increase Acetonitrile to 60%.
Critical Considerations for Optimization
To achieve the "good separation" requested in the prompt, consider these factors if the initial run does not resolve SHR8008X from SHR8008JA:
1. pH Control is Key
Oteseconazole contains basic nitrogen atoms (tetrazole/pyridine moieties).
If peaks are tailing: Ensure your buffer pH is roughly 3.0. This keeps the basic nitrogens protonated and prevents them from interacting with unreacted silanols on the column stationary phase.
Buffer Choice: If you are using Mass Spectrometry (LC-MS), swap the Phosphoric Acid for 0.1% Formic Acid or Ammonium Formate.
2. Modifying Selectivity
If the impurities (SHR8008X and JA) co-elute with the main peak:
Change the Organic Modifier: Substitute Methanol for Acetonitrile. Methanol has different selectivity (protic solvent) compared to Acetonitrile (aprotic) and often resolves positional isomers better.
Change the Column Chemistry: If a standard C18 fails, try a Phenyl-Hexyl column. The pi-pi interactions from the phenyl column often help separate aromatic compounds like azole antifungals better than a standard alkyl chain.
3. Sample Diluent
Dissolve your sample in a mixture of Water:Acetonitrile (50:50). Avoid dissolving in 100% Acetonitrile if your starting gradient conditions are high aqueous, as this causes peak distortion (fronting).
