| Summary: | Cotinine is the major metabolite of nicotine and, being
very stable and having a long biological half-life, it
can be used as a biomarker for tobacco exposure. The
aim of this study was to develop an analytical GC-MS
technique to measure levels of cotinine in the urine of
active and passive smokers and to compare the results
with reference values. The extraction of cotinine to
generate the calibration curve was performed by mixing
urine (250 µL) with 50 µL of a cotinine standard, 50
µL of an internal standard of deuterated cotinine (15
µg∙mL-1) and 50 µL of 10% NH4
OH solution. Next, 2
mL of a mixture of MTBE:dichloromethane:ethyl
acetate (30:30:40 by volume) was added and the whole
was vortexed, then centrifuged at 3000 rpm. Finally, 1.6
mL of the organic layer was evaporated under a stream
of dry air at 50 °C. The resulting extract was dissolved
in methanol and injected into the GC-MS system. The
LOQ and LOD for cotinine were 100 and 20 ng∙mL-1,
respectively. The curve was linear over the whole tested
range of 100 - 5000 ng∙mL-1 and the method achieved
50% recovery. The intra and inter-day precisions were
1.62 – 7.28% and 0.86 – 2.68%, respectively. Accuracy
was determined at three concentrations (low, medium
and high), with six replicates (95.24 – 97.67%). The
validation of this cotinine assay by GC-MS showed
that it exhibited satisfactory limits and the assay could
be performed with a one-step liquid-liquid extraction.
The technique presented here can thus be used for the
quantitation of cotinine levels in the urine of passive
and active smokers.
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