Cotinine/Nicotine MRM Method

Measured Analytes

  1. Nicotine
  2. Cotinine


Liquid chromatography tandem mass spectrometry (LC-MS) was carried out with a Shimadzu Prominence HPLC system and SCIEX 4000 mass spectrometer. An aliquot of 10 µl of each sample was injected onto an Accucore 2.6 µm C18 100 x 3.0 mm column at 40oC for gradient separation. Mobile phases were: A, ddH2O with 0.1% Formic Acid; and B, Acetonitrile with 0.1% Formic Acid. Gradient schedule started at 10% B and was linearly increased to 85% B at 5 minute. At 5.1 minutes the gradient was returned to starting conditions and allowed to re-equilibrate until 8.0 minutes. Flow rate was 400µL/min.


Sample Processing

  1. Extraction Protocols:
    100 µL serum was transferred to a 1.5mL tube containing 25µL trichloroacetic acid, 75µL of water, and 10µL of 250 ng/mL internal standard. Samples were vortexed for 15 minutes then centrifuged at 16,000 x g for 10 minutes. Sola SCX 10mg/1 mL SPE cartridges were activated with 1mL methanol and followed by 1 mL 20 mM ammonium acetate. Sample supernatant was loaded on to the SPE cartridge then washed with 1 mL 20 mM ammonium acetate and 1 mL 30% methanol. Waste tubes were replaced by collection tubes and then the sample was eluted with 1 mL 1.5% NH4OH in methanol. Samples were dried under a gentle stream of N2 gas. Samples were reconstituted in 100µL of 90% 20 mM ammonium bicarbonate/10% acetonitrile, vortexed for 30 seconds and then transferred to LC vial for analysis.




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