It`s Teatime!
Determination of pyrrolizidine alkaloids in tea by means of HPLC-MS/MS after
previous SPE

It's Teatime - a British tea culture that is known and appreciated across the borders of the United Kingdom. As with all foods, it is important that the boiled tea does not contain any unwanted substances. Unlike pesticides, most consumers are not aware of the health risks of contaminants of natural origin. In addition to pesticide analysis, an analysis of natural contaminants such as pyrrolizidine alkaloids is therefore useful for consumer protection.

  The following method which describes the determination of pyrrolizidine alkaloids in tea was kindly made available to us by Ms. Franziska Chmelka from the Department of Technical Food and Environmental Analysis (TeLA GmbH).

  Pyrrolizidine alkaloids are molecules, which contain the bicyclic tertiary amine pyrrolizidine. Of course, these alkaloids are found worldwide as plant constituents in over 6000 different types of flowering plants (for example, composite plants, borage family plants, legumes, orchids) [1]. The common ragwort (Jacobaea vulgaris, also known as Senecio jacobaea), which belongs to the family of the composite plants, contains toxic pyrrolizidine alkaloids. It is widely spread in field’s edges, arable land and meadows, and is not much appreciated by farmers, since all plant parts contain the liver-toxic alkaloids [2]. In contrast to many other poisons, the pyrrolizidine alkaloids remain active in the hay, and are thus also ingested from grazing animals, who would otherwise stay away from the fresh bitter-tasting herbs.

Pyrrolizidine alkaloids can enter the human food chain through plant food. The transition of pyrrolizidine alkaloids into the nectar and with it in honey was demonstrated [3]. Moreover contamination of rocket salad and other salad mixes with Jacobaea vulgaris leafs has been reported. The BfR (Federal Institute for Risk Assessment, Germany) had warned already against  too high pyrrolizidine alkaloids concentrations in herbal teas in 2013 and launched an analytical method [4]. An HPLC-MS/MS method with preceding SPE is presented below.

  HPLC method for the determination of pyrrolizidine alkaloids
  An HPLC-MS/MS method was developed for the determination of the pyrrolizidine alkaloids using a standard mixture of seventeen alkaloids. The separation is successfully achieved by using the 250 x 2 mm column NUCLEODUR® C18 HTec following the chromatographic conditions listed in table 1.

  Table 1: Technical data of the HPLC column and optimized conditions for HPLC analysis

Technical data of the HPLC column and optimized conditions
 HPLC column:
EC 250/2 NUCLEODUR® C18 HTec, 5 µm (REF 760316.20)
 Eluent A: 5 mM formic acid
 Eluent B: methanol
5 % B for 3 min, 5–20 % B in 4 min, 20 % B for 6 min,
20–65 % B in 3 min, 65–95 % B in 1 min, 95 % B for 3 min,
95–5 % B in 0.1 min
 Flow rate: 0.25 mL/min
 Column temperature: 28 °C
 Injection volume:
5 µL
 Detection: MS/MS (MRM, ESI positive, for further parameter and the
chromatogram of standard mixture see MN application no. 127970)

  SPE method for the extraction of pyrrolizidine alkaloids
  A solid phase extraction method was developed for the extraction of alkaloids from leafs and flowers in dried Jacobaea vulgaris. The SPE was carried out with the nonpolar, endcapped C18 phase CHROMABOND® C18 ec under the conditions described in table 2.

  Table 2: Technical data of the SPE column and optimized conditions for SPE method

Technical data of the SPE column and optimized conditions
 SPE column:
CHROMABOND® C18 ec, 3 mL, 500 mg (REF 730013)
 Column conditioning: 5 mL methanol, then 5 mL water
(Do not let run the column dry!)
 Sample aspiration:
5 mL sample (vacuum)
5 mL methanol
 Evaporation: Eluat is boiled down
 Reconstitution: 1 mL 10 % methanol

  Content of pyrrolizidine alkaloids in dried Jacobaea vulgaris
  With the developed methods, seven pyrrolizidine alkaloids were found in leafs and flowers of Jacobaea vulgaris. Table 3 shows the mean recovery rates of the alkaloids from five samples processed in parallel.

  Table 3: Recovery rates in leafs of Jacobaea vulgaris

Pyrrolizidine alkaloids

Average recovery rates [%]
 Erucifoline N-oxide 90
 Jacobine N-oxide
 Senecionine 98
 Retrorsine N-oxide
 Senecionine N-oxide

  Figures 1 and 2 show HPLC-MS/MS chromatograms for the determination of the content of the seven detected pyrrolizidine alkaloids in dried Jacobaea vulgaris.


  Figure 1: Chromatogram of the pyrrolizidine alkaloids in leafs of the Jacobaea vulgaris


  Figure 2: Chromatogram of the pyrrolizidine alkaloids in flowers of the Jacobaea vulgaris

  Contents are determined from 0.858 mg/kg (leafs) and 0.765 mg/kg (flowers) for retrorsine up to 918.000 mg/kg (leafs) and 271.000 mg/kg (flowers) for jacobine N-oxide, respectively figure 3).


  Figure 3: Content of pyrrolizidine alkaloids in dried Jacobaea vulgaris

  Detection of pyrrolizidine alkaloids in chamomile tea
  By the addition of dried Jacobaea vulgaris flowers in commercial chamomile tea, the question "How much pyrrolizidine alkaloids are extracted from boiling water into the tea?" was investigated.

Four series of experiments were carried out:
  • Chamomile without admixture
  • Chamomile with 0.1 % Jacobaea vulgaris flowers
  • Chamomile with 1.0 % Jacobaea vulgaris flowers
  • Chamomile with 10 % Jacobaea vulgaris flowers
Fortunately, no pyrrolizidine alkaloids could be detected in the investigated chamomile tea without an admixture of Jacobaea vulgaris flowers. However according to the high natural content of jacobine N-oxide in Jacobaea vulgaris 0.058 mg/kg (Chamomile + 0.1 % Jv), 0.51 mg/kg (Chamomile + 1.0 % Jv) and 4.18 mg/kg (Chamomile + 10 % Jv) high concentrations in the infusion were found.

On the other hand, retrosine has the lowest values (0.0011 mg/kg, 0.010 mg/kg, 0.091 mg/kg) in the infusion, although the content of erucifoline N-oxide in flowers with 5.060 mg/kg is significantly higher than for retrosine with only 0.765 mg/kg, comparatively low values of 0.0021 mg/kg, 0.0217 mg/kg, and 0.264 mg/kg could be demonstrated (see figure 4).


  Figure 4: Detection of pyrrolizidine alkaloids in chamomile tea

  The analytical method developed by TeLA GmbH with MACHEREY-NAGEL products ensures that drinking tea remains a harmless and delicious consumption before, during and after “Teatime”.

  [1] Pyrrolizidinalkaloide, Wikipedia, https://de.wikipedia.org/wiki/Pyrrolizidinalkaloide (20.09.2017)
[2] Jakobs-Greiskraut, Wikipedia, https://de.wikipedia.org/wiki/Jakobs-Greiskraut (18.09.2017)
[3] R. Hegnauer, Chemotaxonomie der Pflanzen, Birkhäuser, Basel 1989, 281
[4] Gehalte an Pyrrolizidinalkaloiden in Kräutertees und Tees sind zu hoch, Bundesinstitut für Risikobewertung, 18/2013

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The method described was published with the approval of the TeLA GmbH (Geestland, Germany), who is responsible for the correctness of the method. The present article on the method has been edited by MACHEREY-NAGEL. MN cannot assume any guarantee or warranty regarding the selection, efficiency or functionality of the products and services used.

Photo credits: © #40113021, www.fotolia.com

Further applications for the determination of pyrrolizidine alkaloids can be found with MN application nos. 127480 and 127490!

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