Effect of Sample Diluents on the Quantitation of Basic Compounds by High Performance Liquid Chromaography

The detrimental effect of the interaction between basic analytes and silanol groups on the stationary phases during high-performance liquid chromatography (HPLC) separations has been well characterized, and many new stationary phases have been developed to minimize this effect. Similar interactions can happen between basic analytes and silanol groups on glassware surfaces during sample preparation but are often neglected. In this study, we used amitriptyline and its impurity, nortriptyline, as an example to study the effects of sample diluents on their quantitation by HPLC with four different types of glass HPLC vials. Three diluents were studied including water, 100 mM sodium chloride, and 0.1% trifluoroacetic acid. The method performance was evaluated in terms of linearity, precision and accuracy. Low sensitivity and high variability was observed for sample solutions prepared in water, which can be attributed to random adsorption of basic analytes onto the silanol groups of the glassware surfaces. Addition of sodium chloride to increase the ionic strength improved the method performance for both assay and impurity level measurements but did not completely eliminate the interaction with silanols. The most accurate and precise results across all concentration ranges regardless of HPLC vial type were obtained using 0.1% trifluoroacetic acid as the diluent. Graphical Abstract