Simultaneous determination of caffeine, theobromine, theophilline, paraxanthine and nicotine in human milk by liquid chromatography with diode array UV detection

A liquid chromatography-diode array UV detection (LC–UVDAD) method for the simultaneous determination of the alkaloids Nicotine, Caffeine, Theobromine, Paraxanthine and Theophylline is described. The chromatographic separation was achieved on a LC-18-DB column using 20:80 methanol:buffer (5 mM sodium octane sulphonate, 10 mM citric acid adjusted to pH 5.8 with triethylamine) as mobile phase. The method has been applied to human milk samples. The overall procedure had % recoveries ranging from 60.2 ± 0.6 (theobromine) to 98.6 ± 0.4 (caffeine). The within-day (n = 5) and between-days (n = 5 over 5 days) coefficients of variation in milk ranged from 3.7% (caffeine) to 4.7% (theobromine) and from 5.1% (caffeine) to 6.7% (theobromine). Estimated LOD and LOQ in milk ranged from 8 (caffeine) to 13 (nicotine) ng/ml and from 24 (caffeine) to 34 (nicotine) ng/ml, respectively. Samples naturally contaminated with the target analytes were found.