Anal. Chem. 2005, 77, 2421-2425
Determination of Colchicine Residues in Sheep Serum and Milk Using High-Performance Liquid Chromatography Combined with Electrospray Ionization Ion Trap Tandem Mass Spectrometry Gerd Hamscher,*,† Beate Priess,† Heinz Nau,† and Edmond Panariti‡
Department of Food Toxicology, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany, and Institute of Veterinary Research, Aleksander Moisiu Street 10, Tirana, Albania
Colchicine is a naturally occurring alkaloid used in human and veterinary medicine. It shows genotoxicity in in vitro and in vivo systems even at low concentrations. Therefore, no ADI has been established, and colchicine has been included in Annex IV of Council Regulation (EEC) No. 2377/90. No abuse of this drug in intensive livestock farming has yet been reported. However, there may be a natural route of entry for this compound into the food chain when Colchicum autumnale is consumed by animals kept outdoors. To address this concern, we developed and validated a highly sensitive and selective quantitative LC-ESI-MS-MS method for the detection of colchicine in sheep serum and milk. For sample pretreatment, all samples were liquid-liquid extracted with phosphate buffer (pH 8.0) and dichloromethane. LC separation was carried out on an RP C18 column employing a 0.5% formic acid/acetonitrile gradient system. The recoveries in both matrixes at a concentration range from 0.0005 to 1 mg/L were >80% with RSDs of 0.999 for the MS-MS procedure. All injections were performed by employing the push-loop injection mode of the autosampler. The injection volume was generally 8 µL. Milk or serum samples containing colchicine in amounts beyond the linear calibration range were reinjected in volumes down to 1 µL or in a volume of 8 µL after dilution with methanol. We used the MS2 scan mode for quantitative analysis. Quantification was obtained by comparing the peak areas of the sample with that of the external standard. The sum of nine monitored MS-MS ions was used (see Figure 2B). All data were corrected for recovery. The criteria for the confirmation of peak identity were according to Decision 2002/657/EU:13 retention time (RSD 50%, (25% for a relative intensity between 20% and 50%, (30% for a relative intensity between 10% and 20%, and (50% for a relative intensity 50%, (25% for a relative intensity between 20% and 50%, (30% for a relative intensity between 10% and 20%, and (50% for a relative intensity of 0.999, Table 1) over this concentration range. From these data, the limit of quantification of 0.0005 mg/L was established for this method in milk and serum. The smallest amount that could be detected based on a signal-to-noise ratio greater than 5 was ∼0.0001 mg/L. This corresponds to ∼2.4 pg of colchicine on-column, which is at least four times more sensitive than that of all previously published analytical methods,1-9,14,15 In addition, the unequivocal confirmation of colchicine residues can be obtained via MS-MS spectra, which provide at least four significant diagnostic ions (m/z 382, 358, 341, and 326; see Figure (14) Li, W.; Sun, Y.; Fitzloff, J. F.; van Breemen, R. B. Chem. Res. Toxicol. 2002, 15, 1174-1178. (15) Panariti, E. Dtsch. Tierarztl. Wochenschr. 1996, 103, 128-129.
2424 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005
Table 2. Results Obtained for 10 Sheep Serum Samples and the Corresponding Milk Samples Analyzed for Colchicine animal no.
serum (mg/L)
milk (mg/L)
1 2 3 4 5 6 7 8 9 10
0.79
