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).

Keywords: HPLC-QQQ-MS/MS chaotropic effect; RP-HPLC-DAD; cotinine; nicotine; trans-3′-hydroxycotinine.

Figure 1

HPLC-DAD chromatogram obtained for solution of standards: trans-3′-hydroxycotinine (tr = 4.63 min), cotinine (tr = 6.16 min), nicotine (tr = 11.01 min) at the concentration level of 500 ng L⁻¹. The analysis was performed using an Agilent 5 HC-C18(2) (250 × 4.6 mm I.D.) column. The mobile phase was acetonitrile (10%, v/v), 20 mM phosphate buffer pH = 2.7 containing 30 mM NaPF₆ in the whole mobile phase. The DAD detection was set at 260 nm.

Figure 2

UV-absorption spectra of nicotine, cotinine, and trans-3′-hydroxycotinine standards measured from 220 to 400 nm.

Figure 3

Chromatogram of a mixture of standards with a concentration of 2 mg mL⁻¹ dissolved in methanol (black line) and in the mobile phase (red line). The detection limits [ng mL⁻¹] were estimated considering the analyte concentration that produces a chromatographic peak having a height equal to three times the standard deviation of the baseline noise.

Figure 4

Representative chromatograms of blank plasma sample (a) and plasma spiked with trans-3′-hydroxycotinine, cotinine, nicotine standards at a concentration of 1000 ng L⁻¹ (b), 500 ng L⁻¹ (c), and 100 ng L⁻¹ (d).

Figure 4

Representative chromatograms of blank plasma sample (a) and plasma spiked with trans-3′-hydroxycotinine, cotinine, nicotine standards at a concentration of 1000 ng L⁻¹ (b), 500 ng L⁻¹ (c), and 100 ng L⁻¹ (d).

Figure 5

HPLC-QQQ-MS/MS chromatograms of quantitative transition of nicotine (a) and metabolites: cotinine (b), trans-3′-hydroksycotinine (c) from the blank plasma sample, plasma samples used in the validation process and smoker’ plasma sample. Plasma sample spiked with 100 ng mL⁻¹ of analytes (blue); Plasma sample spiked with 20 ng mL⁻¹ of analytes (green); smoker’ plasma sample (red); blank plasma sample (black).

Figure 5

HPLC-QQQ-MS/MS chromatograms of quantitative transition of nicotine (a) and metabolites: cotinine (b), trans-3′-hydroksycotinine (c) from the blank plasma sample, plasma samples used in the validation process and smoker’ plasma sample. Plasma sample spiked with 100 ng mL⁻¹ of analytes (blue); Plasma sample spiked with 20 ng mL⁻¹ of analytes (green); smoker’ plasma sample (red); blank plasma sample (black).

Figure 6

HPLC-QQQ-MS/MS chromatograms of quantitative (black) and confirmatory (blue) transitions of nicotine and metabolites from the blank plasma sample (a), plasma samples used in the validation process (b), and smoker’ plasma sample (c).