A quantitative method using ultra performance liquid chromatography–tandem mass spectrometry is described for simultaneous determination of nicotine and its metabolites (cotinine and trans‐3′‐ hydroxycotinine) in human plasma. Aliquots of 0.25 mL of plasma specimens were used for analysis, and 3 analytes were extracted by liquid–liquid extraction. The main problem was blank plasma contamination with environmental nicotine. Activated charcoal was used to avoid this analytical interference. For optimized chromatographic performance, a basic mobile phase consisting of 0.2% ammonia in water (mobile phase A, pH10.6) and acetonitrile (mobile phase B) was selected. The analytes were separated on a 50 mm × 2.1 mm BEH C18 column, 1.7 μm particle size, and quantified by MS/MS using multiple‐reaction monitoring (MRM) in positive mode. The chromatographic separation was achieved in 3 min followed by 1.2 min of column equilibration. The calibration curves were linear in the concentration range of 10–1000 ng/mL with correlation coefficients exceeding 0.99. Within‐day precisions and between‐day precisions (CV, %) were <15 %. The accuracy expressed as bias was within ±15% for all analytes. The recovery values ranged from 50% to 97%. The ions used for quantification of nicotine, cotinine and 3‐OH‐cotinine were 166.9 > 129.7; 176.9 > 79.7; 192.9 > 79.7 m/z, respectively. The original blank sample preparation solved the problem of contamination in a cost‐effective and efficient way. The validated method has been routinely used for analysis of nicotine and metabolites and determination of hydroxycotinine/cotinine metabolic ratio. This biomarker seems to be interesting at predicting response of nicotine patch replacement therapies.
Fundamental & Clinical Pharmacology – Wiley
Published: Oct 1, 2015