Nicotine and metabolites determination in human plasma by ultra performance liquid chromatography-tandem mass spectrometry: a simple approach for solving contamination problem and clinical application.

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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.