A sensitive LC-MS/MS method for isomer separation and quantitative determination of 51 pyrrolizidine alkaloids and two tropane alkaloids in cow's milk

Abstract

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid-liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSD_r) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected.

Keywords: Liquid chromatography tandem mass spectrometry; Method development; Milk; Pyrrolizidine alkaloids; Tropane alkaloids.

Fig. 1

Examples of pyrrolizidine alkaloids, their corresponding N-oxides and tropane alkaloids, representing different core structures and grades of esterification

Fig. 2

Box and whisker plot of the precision, expressed as the relative standard deviation (RSD), of extraction and clean-up procedures using C18 cartridges with n-hexane (n = 3) and polymer cation exchange (PCX) cartridges with and without n-hexane (n = 4). Replicates were spiked to a concentration of 12.3 ng/mL in the final measuring solution. The box corresponds to the range in which the middle 50% of the data are located. Whiskers mark the 1.5 interquartile range. Circles indicate individual outliers outside the 1.5 interquartile range. Squares represent the mean and lines the median

Fig. 3

LC–MS/MS chromatograms of milk samples artificially contaminated with 30 pyrrolizidine alkaloids, 24 pyrrolizidine alkaloid N-oxides, and two tropane alkaloids to concentrations of 0.50 µg/L (a) and 0.05 µg/L (b) per individual analyte, prepared and measured using the final method. For abbreviations of the analytes, see Table 1