Measurement of nicotine and cotinine in human milk by high-performance liquid chromatography with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) assay for the determination of nicotine and cotinine in human milk was developed using an extraction by liquid–liquid partition combined with back extraction into acid, and followed by reverse-phase chromatography with UV detection of analytes. The assay was linear up to 500 μg/l for both nicotine and cotinine. Intra- and inter-day relative standard deviations (R.S.D.) were <10% (25–500 μg/l) for both nicotine and cotinine. Limits of quantitation (LOQ) were 10 and 12 μg/l for nicotine and cotinine, respectively, while the limits of detection (LOD) were 8 and 10 μg/l for nicotine and cotinine, respectively. The mean recoveries were 79–93% (range 25–500 μg/l) for nicotine and 78–89% (range 25–500 μg/l) for cotinine. The amount of fat in the milk did not affect the recovery. We found that this method was sensitive and reliable in measuring nicotine and cotinine concentrations in milk from a nursing mother who participated in a trial of the nicotine patch for smoking cessation.