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

Abstract1,2-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 (RSDr) 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. Graphical abstract

QuEChERS method combined to liquid chromatography high‐resolution mass spectrometry for the accurate and sensitive simultaneous determination of pyrrolizidine and tropane alkaloids in cereals and spices

“…15,22,24,31,32,34 HPLC resolution is crucial for the successful identification and differentiation of isomeric monoesters intermedine and lycopsamine (m/z 300) and their N-oxides (m/z 316) as well as the cyclic diesters senecionine and senecivernine (m/z 336) and their N-oxides (m/z 352), among the PAs/PANOs recommended by EFSA to be monitored in food. To date, according to the literature, many authors present the phenomenon of co-elution (as highlighted above), other, instead of including these eight isomers, only analyse some of them 33,38 or apply different acidic and alkaline mobile phases 39 (MPs) or try to separate the highly polar alkaloids using hydrophilic interaction liquid chromatography (HILIC) columns. 24,39,40 As far we know, there are only three papers able to obtain the complete separation of all the PAs/PANOs recommended by EFSA including the isomers.…”

“…To date, according to the literature, many authors present the phenomenon of co-elution (as highlighted above), other, instead of including these eight isomers, only analyse some of them 33,38 or apply different acidic and alkaline mobile phases 39 (MPs) or try to separate the highly polar alkaloids using hydrophilic interaction liquid chromatography (HILIC) columns. 24,39,40 As far we know, there are only three papers able to obtain the complete separation of all the PAs/PANOs recommended by EFSA including the isomers. Kaltner et al 29 and Dreolin et al 32 focused their attention mainly on PAs in teas and herbal teas and plant-based food and honey, respectively.…”

Pyrrolizidine Alkaloid Extraction and Analysis: Recent Updates

“…There are some PA isomers recommended to be monitored by the European Commission Regulation 2020/2040, e.g., indicine, echinatine, rinderine (possible coelution with lycopsamine/intermedine), indicine- N -oxide, echinatine- N -oxide, rinderine- N -oxide (possible coelution with lycopsamine- N -oxide/intermedine- N -oxide), integerrimine (possible coelution with senecivernine/senecionine), integerrimine- N -oxide (possible coelution with senecivernine- N -oxide/senecionine- N -oxide), heliosupine (possible coelution with echimidine), heliosupine- N -oxide (possible coelution with echimidine- N -oxide), spartioidine (possible coelution with seneciphylline), spartioidine- N -oxide (possible coelution with seneciphylline- N -oxide), usaramine (possible coelution with retrorsine), and usaramine N -oxide (possible coelution with retrorsine N -oxide) [ 47 ]. Chromatographic resolution is fundamental for the differentiation of isomeric PAs such as intermedine, indicine, lycopsamine, rinderine, and echinatine ( m / z 300) and their N -oxides ( m / z 316) as well as integerrimine, senecionine, and senecivernine ( m / z 336) and their N -oxides ( m / z 352), [ 100 ]. Klein, et al [ 100 ] applied different acidic and alkaline mobile phases and succeeded to differentiate between some of the PA isomers, especially when alkaline conditions were applied.…”

“…Chromatographic resolution is fundamental for the differentiation of isomeric PAs such as intermedine, indicine, lycopsamine, rinderine, and echinatine ( m / z 300) and their N -oxides ( m / z 316) as well as integerrimine, senecionine, and senecivernine ( m / z 336) and their N -oxides ( m / z 352), [ 100 ]. Klein, et al [ 100 ] applied different acidic and alkaline mobile phases and succeeded to differentiate between some of the PA isomers, especially when alkaline conditions were applied. In the same study, the dimension of the C 18 column and its particle size affected the resolution of the PA peaks produced.…”

Strukturmodifikationen bestimmter Pyrrolizidinalkaloide während der Probenextraktion in analytischen Methoden

“…Als mögliche Ursache wurden Umwandlungsreaktionen wie Deacetylierung [1] bzw. Epoxidbildung [2] aufgrund von Bedingungen ähnlich zur sog. Chlorohydrin-Reaktion bei der Herstellung von Epoxiden [3]…”