Measurement of cotinine in urine by liquid chromatography tandem mass spectrometry

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Measurement of cotinine in urine by liquid
chromatography tandem mass spectrometry

• C A Chadwick, B G Keevil

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Outline

• Cotinine
• Methods
• Chromatography
• Mass spectrometry
• Ion suppression
• Validation
• Stability

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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.

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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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Methods

• Colorimetric method
• Immunoassay
• ELISA
• Gas/liquid chromatography MS/MS

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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.

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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.

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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.

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Chromatogram

• A d3-cotinine
• RT 2.5 minutes
• B Cotinine 50 µg/L
• RT2.5 minutes

A
B
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Mass spectrometry
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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.

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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 (gt10 ) in
  the specific signal for cotinine.

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Ion Suppression hydro
cotinine
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Ion suppression SCX
cotinine
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Ion suppression SCX and hydro
cotinine
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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.

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Ion Suppression
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Linearity
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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.

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Imprecision
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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.

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Stability

• Extracted QC samples over a 24 hr period.
• Response at t24 compared with response at t0.
• Decrease in response was 4 at all QC
  concentrations

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Room Temperature Stability
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Fridge Stability
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Freezer stability
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Stability

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

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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

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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.