Figure 2: Setup of the
SFC-MS/MS analytical system
tivity and robustness. However, this is only possible,
when the BPR volume is low enough not to cause any
peak dispersion in the flow line.
The tea samples were extracted twice with 0.05 M sulfuric
acid by sonication, followed by centrifugation.
The pH of the combined extracts was neutralized with
ammonium hydroxide before the samples were subjected
to a solid phase extraction (SPE) 4. After elution,
the extracts were dried in a stream of nitrogen at 50 °C
for about 60 minutes. The samples were reconstituted
with 1 mL methanol, mixed thoroughly and centrifuged
for 10 min before transfer to SFC-MS analysis.
Analytical conditions are summarized in Table 1.
SFC-MS/MS method development
The main mobile phase used for SFC is supercritical
carbon dioxide, to which polar organic solvents (mod-
Table 1: Analytical conditions
Figure 3: Chromatogram of the SFC-MS analysis of 34
ifiers) are added for control of solubility and polarity.
Ionic additives in aqueous or organic solution can also
be used to adjust selectivity. Method scouting for the
34 different PAs was performed by testing 32 combinations
of stationary and mobile phases (a set of four different
columns from the Daicel CHIRALPAK® series and
eight different modifiers). After identification of the
most suitable combination for separation of the 34 PAs,
gradient conditions, flow rate and column temperature
were optimized to achieve baseline separation of all isomers
in a minimized overall analysis time (Figure 3).
Analysis of tea samples
Using the reported instrument set-up, quantification
of 18 PAs and 16 of their related N-Oxides could be
achieved. Calibration curves in black tea matrix (determined
in duplicate) showed good precision and accuracy.
Even in a complex matrix like tea, it was possible
to quantify the PAs easily in the range of at least
2 to 200 μg/kg. For all analytes, weighted regression
resulting in r² > 0.99 was obtained, with S/N > 10
for LLOQ levels. Exemplary calibration curves are displayed
in Figure 4.
In an application example, 10 commercially available
tea samples were analyzed. In 4 out of the 10 samples,
one or more pyrrolizidine alkaloids could be detected
above their LLOQ. Europine, Heliotrine, Lasiocarpine
and their related N-oxides were present in one sample,
Figure 4: Calibration curves in black tea matrix
Figure 5: Chromatograms of the separation of Lycopsamin
and Senecionine isomers