Two ml of normal urine containing standard
cotinine ranging from 1 to 100 microgram/ml, urine
samples, quality control samples, and blank urine
samples were placed into 10-ml glass stopper test tubes,
adjusted to pH 9 using 10.0% sodium carbonate and
10.0 microlitre of diphenylamine (10 mg/l of methanol)
as internal standard were added. The solution was
extracted with 5.0 ml of dichloromethane, centrifuged,
and the lower layer of dichloromethane was kept to
evaporate under nitrogen blow until dryness.