In the process of analysis and testing, compounds with higher polarity are usually poorly retained on conventional non-polar columns. Even if the organic phase ratio is reduced to a very low level, it is difficult to achieve retention enhancement, which is easily interfered by blank solvents or blank excipients. Therefore, eliminating interference has become a very important part of method setting.


Aminobutyramide has high polarity, weak UV absorption, almost no retention on conventional C18 column, and the blank of auxiliary material interferes with its determination.

The structure is shown in the figure:

Original condition

Column: Ultisil® AQ-C18 (4.6×250mm, 5μm).

Mobile phase: phosphate buffer: acetonitrile = 95:5;


The target peak is polar and has no retention on the C18 column. The peak appears in about 3 minutes, which coincides with the blank of the auxiliary material and cannot be separated.

blue – excipient peak black – target

Method development ideas

To separate the target substance from the excipients, the retention of the target compound must be enhanced. There are two amino groups on the structure of this substance, and the mode determination of adding ion-pairing reagent in the mobile phase can be considered. Combined with its detection wavelength (205nm), sodium dodecyl sulfate can be added to the mobile phase system for determination, and adjusted by the method Optimization can eliminate the interference of blank excipients on impurities.

Adjusted chromatographic conditions

Column: Ultisil® XB-C18 (4.6×250mm, 5μm).

Mobile phase: 0.01mol/L potassium dihydrogen phosphate+0.5g/L sodium dodecyl sulfate (pH2.5)/acetonitrile=70/30.


As expected, the retention of the target compound was significantly enhanced in this system, which was not affected by the blank of excipients, and better retention and separation could be achieved.

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