Application of HPLC-QQQ-MS/MS and New RP-HPLC-DAD System Utilizing the Chaotropic Effect for Determination of Nicotine and Its Major Metabolites Cotinine, and trans-3′-Hydroxycotinine in Human Plasma Samples

Abstract

The routine techniques currently applied for the determination of nicotine and its major metabolites, cotinine, and trans-3′-hydroxycotinine, in biological fluids, include spectrophotometric, immunoassays, and chromatographic techniques. The aim of this study was to develop, and compare two new chromatographic methods high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS), and RP-HPLC enriched with chaotropic additives, which would allow reliable confirmation of tobacco smoke exposure in toxicological and epidemiological studies. The concentrations of analytes were determined in human plasma as the sample matrix. The methods were compared in terms of the linearity, accuracy, repeatability, detection and quantification limits (LOD and LOQ), and recovery. The obtained validation parameters met the ICH requirements for both proposed procedures. However, the limits of detection (LOD) were much better for HPLC-QQQ-MS/MS (0.07 ng mL−1 for trans-3′-hydroxcotinine; 0.02 ng mL−1 for cotinine; 0.04 ng mL−1 for nicotine) in comparison to the RP-HPLC-DAD enriched with chaotropic additives (1.47 ng mL−1 for trans-3′-hydroxcotinine; 1.59 ng mL−1 for cotinine; 1.50 ng mL−1 for nicotine). The extraction efficiency (%) was concentration-dependent and ranged between 96.66% and 99.39% for RP-HPLC-DAD and 76.8% to 96.4% for HPLC-QQQ-MS/MS. The usefulness of the elaborated analytical methods was checked on the example of the analysis of a blood sample taken from a tobacco smoker. The nicotine, cotinine, and trans-3′-hydroxycotinine contents in the smoker’s plasma quantified by the RP-HPLC-DAD method differed from the values measured by the HPLC-QQQ-MS/MS. However, the relative errors of measurements were smaller than 10% (6.80%, 6.72%, 2.04% respectively).

Application of Ionic Liquids as Mobile Phase Additives for Simultaneous Analysis of Nicotine and Its Metabolite Cotinine in Human Plasma by HPLC–DAD

“…Both are strong bases, which means they were in their cationic state at the working pH of 2.3. In the chemical structure of nicotine there is a pyridine ring (pKa 2.96) and a pyrrolidine ring (pKa 8.5), so that in an acidic aqueous medium, it appears as a diprotonated cation with a charge on both the ionizable fragments [ 32 ]. Since in cotinine’s structure there is a conversion of the cyclic amino group into a gamma lactam nitrogen structure, the pyrrolidine ring lost its basicity in this study.…”

Development in competitive immunoassay of a point-of-care testing for cotinine (COT) detection in urine

“…24 These methods are highly specic, but they come with challenges in terms of complexity and cost, requiring the expertise of specialists. 22 Although radioimmunoassay (RIA) is also sensitive and specic, it generates toxic and carcinogenic waste products, limiting its practicality. 25 Current methods oen involve complex laboratory procedures, lengthy processing times, and specialized equipment, limiting their practicality for rapid, on-site testing scenarios.…”

“…20,21 However, currently the detection of COT is primarily performed in research settings rather than routine examinations. Various techniques are used for this purpose, including high-performance liquid chromatography (HPLC), 22 gas–liquid chromatography-mass spectrometry (GLC-MS) 23 and an enzyme-linked immunoassay (ELISA). 24 These methods are highly specific, but they come with challenges in terms of complexity and cost, requiring the expertise of specialists.…”

Simple HPLC method for simultaneous quantification of nicotine and cotinine levels in rat plasma after exposure to two different tobacco products

“…Yet, higher operational cost is still one of the main limitations of their use. HPLC-PDA can provide good sensitivity, rapid analysis and low running costs [41,42], and has been employed efficiently for simultaneous determination of nicotine and cotinine in plasma [43][44][45][46][47][48][49][50][51][52].…”