Setting appropriate cutoff values and the use of a highly sensitive analytical method allow for correct classification of the smoking status. Urine-saliva pairs samples of pregnant women in the second and third trimester, and saliva only in the first trimester were collected. Offline SPE and LC-ESI-MS/MS method was developed in the broad concentration range (saliva 0.4–1000 ng/mL, urine 0.8–4000 ng/mL). The mean recoveries were 3.7 ± 7.6% for urine and 99.1 ± 2.6% for saliva. LOD for saliva was 0.12 ng/mL and for urine 0.05 ng/mL; LOQ was 0.4 ng/mL and 0.8 ng/mL, respectively. Intraday and interday precision equaled, respectively, 1.2% and 3.4% for urine, and 2.3% and 6.4% for saliva. There was a strong correlation between salivary cotinine and the uncorrected cotinine concentration in urine in the second and third trimesters of pregnancy. The cutoff values were established for saliva 12.9 ng/mL and urine 42.3 ng/mL or 53.1 μg/g creatinine with the ROC curve analysis. The developed analytical method was successfully applied to quantify cotinine, and a significant correlation between the urinary and salivary cotinine levels was found. The presented cut-off values for salivary and urinary cotinine ensure a categorization of the smoking status among pregnant women that is more accurate than self-reporting.
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