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Measurement of cotinine in urine by liquid chromatography tandem mass spectrometry

Outline. CotinineMethodsChromatographyMass spectrometryIon suppressionValidationStability. Cotinine. Major metabolite of nicotine.Biomarker for nicotine exposure.Measured in urine, plasma, saliva, hair and semen.Half-life 19 hours.Nicotine metabolism only source of cotinine.. Smokers. Nicotine replacement and smoking cessation.Cotinine measurements are significantly higher in smokers compared to non-smokers.Quantitative method, monitor smoking habits..

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Measurement of cotinine in urine by liquid chromatography tandem mass spectrometry

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    1. Measurement of cotinine in urine by liquid chromatography tandem mass spectrometry C A Chadwick, B G Keevil

    2. Outline Cotinine Methods Chromatography Mass spectrometry Ion suppression Validation Stability

    3. Cotinine Major metabolite of nicotine. Biomarker for nicotine exposure. Measured in urine, plasma, saliva, hair and semen. Half-life 19 hours. Nicotine metabolism only source of cotinine.

    4. Smokers Nicotine replacement and smoking cessation. Cotinine measurements are significantly higher in smokers compared to non-smokers. Quantitative method, monitor smoking habits.

    5. Methods Colorimetric method Immunoassay ELISA Gas/liquid chromatography MS/MS

    6. Implementation Assessment of patients for lung transplantation. Patients who attend chest clinic. Developed and validated a method using reversed phase HPLC coupled to MS/MS.

    7. Sample preparation 10 µL urine sample to 10 µL d3-cotinine (100 µg/L). 100 µL MeCN. Thermo-seal. Vortex- 60 seconds. Centrifuge 5 minutes, 8000 g. Autosampler, 12 µL injected into LC system.

    8. Chromatography Waters TM 2795 Alliance HT LC system. Security guard SCX cation exchange column ( 4.0 x 3.0 mm (i.d.)) and a 30 x 3.0 mm 4 µm Synergi Hydro RP 80 A column. Cotinine captured on SCX column, online solid phase extraction cartridge. Solvent delay for first 0.80 minutes. Cotinine eluted using salt gradient onto analytical column.

    9. Chromatogram A= d3-cotinine RT= 2.5 minutes B= Cotinine 50 µg/L RT=2.5 minutes

    10. Mass spectrometry

    11. Ion Suppression Matrix associated phenomenon caused by compounds that co-elute with the compound of interest and compete for ionisation in the mass spectrometer source.

    12. Ion Suppression 1mg/ml solution of cotinine in MeOH, infused directly into the mass spectrometer. Constant signal in MRM channel for cotinine. Extracted urine sample injected into system via the LC. Ion suppression seen as a reduction (>10 %) in the specific signal for cotinine.

    13. Ion Suppression hydro

    14. Ion suppression SCX

    15. Ion suppression SCX and hydro

    16. Ion Suppression Assessed ion suppression caused by biological matrix. Compared response values of cotinine in water to response values of cotinine at same final concentration added to 5 non-smoker urine samples. Samples run in duplicate.

    17. Ion Suppression

    18. Linearity

    19. Validation Recovery: measure baseline cotinine on 3 urine samples and after addition of cotinine (100, 450, 900 µg/L). Mean recovery 112 %, range (107-117%). Lower limit of quantitation 2.5 µg/L. Lower limit of detection 0.156 µg/L.

    20. Imprecision

    21. Stability Stability of extracted urine sample. Repeated injection every 12 mins for 17 hrs. Cotinine/d3-cotinine peak area ratios (PAR). CV for PAR was 1%.

    22. Stability Extracted QC samples over a 24 hr period. Response at t=24 compared with response at t=0. Decrease in response was 4 % at all QC concentrations

    23. Room Temperature Stability

    24. Fridge Stability

    25. Freezer stability

    26. Stability Urine cotinine analysed on day of collection. Samples should be stored at –20 oC for five days.

    27. Stability 10 individual specimens of human urine, containing cotinine from smokers. Mean of results from 3 replicates expressed as % of baseline values. Moyer T P et al, Clin Chem 2002; 48: 1460-1471

    28. Summary Cotinine specific marker for nicotine exposure. Simple and rapid sample preparation Runtime reduced to four minutes. Novel online ion exchange procedure. SCX and Hydro columns give no significant ion suppression.

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