Cotinine Determination by HPLC RP Separation Coupled to UV Detection Using Ascentis™ C18 Column

By: Pasquale Moio, Andrea Urbani, Reporter EU Volume 22

Pasquale Moio and Andrea Urbani, Centro Studi sull’Invecchiamento (Ce.S.I.), Fondazione Università “G. D’Annunzio”, Chieti, Italy and Dipartimento di Scienze Biomediche, Università “G. D’Annunzio” di Chieti e Pescara, Italy.


Tobacco smoking elicits a complex effect on both central and peripheral nervous system with a deep impact on behaviour. The pharmacological symptoms of the nicotine, a tobacco-derived alkaloid, have been widely investigated. However, relatively little attention has been drawn on possible actions of its major metabolite cotinine. The retention of cotinine in blood and brain after nicotine consumption greatly exceeds that of nicotine. Therefore, cotinine has been suggested as a mediator of the more protracted pharmacological effects of nicotine. We have developed a method for the quantitative determination of cotinine from plasma samples by HPLC using an Ascentis TM C18 column.

back to top

Principles and Methods

Cotinine and 3’-hydroxycotinine (as an internal standard (IS))were extracted from human plasma. The samples were alkalinised in 2 ml polypropylene tubes with 10 μl of 10 mM NaOH and extracted with 1.9 ml of dichloromethane by shaking for 10 min. After centrifugation at 1500 X g for 10 min, the supernatant was discarded and the remaining dichloromethane was transferred to a clean polypropylene tube. Concentrated HCl (20 μl) was added to each sample tube to prevent the volatilisation of nicotine, the samples were gently shaken and evaporated to dryness. Residues were reconstituted in 100 μl of the HPLC mobile phase and 80 μl aliquotes were injected onto the HPLC column.

Separation was performed on an AscentisTM C18, 5 μm, 150 X 4.6mm. The mobile phase A contained CH3CN supplemented with TFA 0.15 % and mobile phase B H20 supplemented with 0.2 % TFA. The mobile phase was delivered at 1 ml/min; the separation gradient is described in Table 1. Analytes of interest were detected by their UV absorbance at 256 nm. In Figure 1 the chromatogram of a plasma extracted cotinine analysis is shown. Both the analyte and the internal standard were well retained and resolved. Quantitative analysis was pursued by plotting the ratio of cotinine to internal standard peak areas versus the cotinine amount. Linear responses (R = 0.998) over a wide quantitative range from 10 ng to 800 ng cotinine were obtained (data not shown).

Table 1.

Figure 1.

back to top


The quantitation of cotinine and other molecular markers for tobacco smoke exposure is currently considered as a valuable tool in forensic medicine and research. We have successfully evaluated Ascentis C18 for quantitation of cotinine from human plasma, implying the applicability of this methodology for accurate cotinine quantitation.

back to top

Related Links