Quantitation of cotinine and its metabolites in rat plasma and brain tissue by hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS)

Abstract

In this work, we developed a sensitive method to quantify cotinine (COT), norcotinine (NCOT), trans-3'-hydroxycotinine (OHCOT) and cotinine-N-oxide (COTNO) in rat plasma and brain tissue, using solid phase extraction (SPE), hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The linear range was 1-100 ng/mL for each analyte in rat plasma and brain homogenate (3-300 ng/g brain tissue). The method was validated with precision within 15% relative standard deviation (RSD) and accuracy within 15% relative error (RE). Stable isotope-labeled internal standards (IS) were used for all the analytes to achieve good reproducibility, minimizing the influence of recovery and matrix effects. This method can be used in future studies to simultaneously determine the concentrations of COT and three major metabolites in rat plasma and brain tissue.

Figure 1

Chemical structures, pKa and XLogP values of COT, NCOT, OHCOT and COTNO. Structures were generated by ChemBioDraw Ultra 12.0 software. pKa and XLogP3 values were obtained from PubChem database.

Figure 2

Representative chromatograms of plasma samples. For each analyte, chromatograms of the analyte and IS were shown for both a spiked standard at LLOQ (1 ng/mL) (A) and a blank sample (B). The concentrations of IS were all 50 ng/mL.

Figure 3

Representative chromatograms of brain homogenate samples. For each analyte, chromatograms of the analyte and IS were shown for both a spiked standard at LLOQ (1 ng/mL) (A) and a blank sample (B). The concentrations of IS were all 50 ng/mL.

Figure 4

Representative chromatograms of plasma samples from rats subcutaneously dosed by 1 mg/kg COT: (A) chromatograms of COT and IS in samples that were diluted 15 folds with blank plasma, with the original COT concentration of ng/mL; (B) chromatograms of NCOT and IS, with NCOT concentration below LLOQ; (C) chromatograms of OHCOT and IS, with OHCOT concentration of ng/mL; (D) chromatograms of COTNO and IS, with COTNO concentration of ng/mL.

Figure 5

Representative chromatograms of brain samples from rats subcutaneously dosed by 1 mg/kg COT: (A) chromatograms of COT and IS in samples that were diluted 15 folds with blank brain homogenate, with the original COT concentration of ng/g; (B) chromatograms of NCOT and IS, with NCOT concentration below LLOQ; (C) chromatograms of OHCOT and IS, with OHCOT concentration below LLOQ; (D) chromatograms of COTNO and IS, with COTNO concentration below LLOQ.