36
QUALITY MANAGEMENT
Determination
of pyrrolizidine
alkaloids in tea
High sensitivity analysis using SFC-MS/MS
›Authors:
Uwe Oppermann1, Gesa Schad1, Anja Grüning1, Jan Stenzler2
1Shimadzu Europa GmbH, Marketing Europe, Analytical BU,
FoodMarket, 47269 Duisburg, Germany
2Shimadzu Deutschland GmbH, 47269 Duisburg, Germany
Pyrrolizidine alkaloids (PAs) are heterocyclic secondary
plant metabolites considered to be carcinogenic
and genotoxic. They occur mainly in plants of the
Boraginaceae, Asteraceae and Fabaceae. Estimates indicate
that about 3 % of the world’s flowering plants,
which is about 6000 species, produce pyrrolizidine alkaloids,
making them one of the most important classes
of naturally occurring toxins.
Long-term exposure to PAs in food, beverages or
phytopharmaceuticals is a possible concern for human
health and, according to the German Federal Institute
for Risk Assessment (BfR) can lead to severe hepatic
damage. So the Panel on Contaminants in the Food
Chain (CON-TAM) have proposed a list of PAs to be
monitored in foodstuffs 1, 2.
PAs display a wide structural diversity, several
hundred already having been identified, with novel
structures being discovered continuously. Pyrrolizidine
alkaloids consist of a necine base typically including
pyrrolizidine esterified with a necic acid. When they
also carry a 1,2-double bond as well as an esterified
side chain, they exhibit hepatotoxic activity 3.
Since some of the analytes are isomers that can’t be
distinguished by different mass, they have to be separated
chromatographically. LC-MS/MS is the standard
method for determination of PAs. However, separation
of these compounds often poses a challenge. SFC (supercritical
fluid chromatography) offers complementary chromatographic
selectivity to RP-LC (reversedphase liquid
chromatography) and an advantage for separation of
stereoisomers,
shown here in the development of a separation
method for determination of 34 PAs including 5
Lycopsamin and 2 Senecionin isomers.
System configuration
The method of choice is supercritical fluid chromatography
using the Nexera UC system coupled with
an LCMS-8060 tandem mass spectrometer (Shimadzu
Corporation, Japan) which has been optimized to obtain
baseline separation of all isomers in the mixture
in just 8 minutes using a Chiralpak IG-3, 100 x 3.0
mm, 3 μm column and a mobile phase modifier consisting
of 50 mM ammonium formate and methanol.
For hyphenation to MS from SFC, a direct transfer
with a heated backpressure regulator (BPR) is most
Figure 1: General structure of pyrrolizidine alkaloids beneficial in terms of stable spray formation, sensi