An accurate and sensitive method for the quantification of 33 pyrrolizidine and 21 tropane alkaloids in plant-based food matrices

Using the SCIEX QTRAP® 6500+ LC-MS/MS System

1Jianru Stahl-Zeng, 2Jack Steed, 1Jasmin Meltretter
1SCIEX, Germany; 2SCIEX, UK

Abstract

Pyrrolizidine and tropane alkaloids have recently become a concern in food aid packaging. Here, an accurate and sensitive method for the quantification of 33 pyrrolizidine and 21 tropane alkaloids in plant-based food matrices has been developed SCIEX QTRAP 6500+ LC-MS/MS System.

RUO-MKT-02-12768-A_f0

Introduction

Recent incidents of poisoning from pyrrolizidine and tropane alkaloids, present in cereals distributed in food aid packages, has heightened awareness regarding the lack of regulations around these naturally occurring toxins. Several scientific opinions have been put forward by EFSA2-6 and maximum residues limits for Europe7 are under consideration. The issue is of such importance that a joint FAO/WHO expert meeting has been held and a special safety evaluation has been produced.8

In anticipation of coming legislation, a sensitive and robust analytical method will be required to meet the increased need for routine testing of plant-based food and commodities. Here, a method has been developed for the detection and quantification of 54 alkaloids in plant-based food matrices using the SCIEX QTRAP 6500+ LC-MS/MS System.1

Figure 1. Chromatographic separation of the 54 alkaloids. The XIC overlays shown here for a 10 ng/mL standard solution highlight the distribution of elution that was possible in the method. This helps to achieve accurate quantification of all 54 alkaloid analytes within a 22-minute run time.

Methods

Chromatography: Chromatographic separation was achieved using an ExionLC™ System with a Phenomenex Luna Omega C18 column with a 1.6 µm particle size. Various mobile phases were tried as detailed by Dzuman et al.1 A final method with a run-time of 22 minutes was developed to give the most efficient resolution with respect to analysis time.

Mass spectrometry: MS analysis was performed on the QTRAP 6500+ System using the IonDrive™ Turbo V Ion Source in electrospray ionization mode. All 54 analytes were optimized to determine compound dependent parameters: entrance potential (EP), declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP). The instrument was operated in positive ion mode with source parameters optimized according to the mobile phase selected in each experiment. See reference 1 for full method details.

 

Separation of isomers

With this method, baseline chromatographic separation was achieved for 49 of the 54 alkaloids (Figure 1). However, even utilizing UHPLC and a slow gradient to maximize separation, reverse phase chromatography is unable to separate five of the compounds due to containing two groups of isomers. This lack of separation has previously been noted in several other publications 9-11, so the upcoming EFSA regulation is expected to allow for certain sums of the most difficult-to-separate isomers to be reported. However, if separation of these compounds is deemed necessary, options such as HILIC chromatography1 or the use of SelexION® Technology, which can be added to the QTRAP 6500+ System, can aid in isomeric separation and has been covered in references 1 (HILIC) and 12 (SelexION Technology). 

Table 1: List of compounds which have been analyzed and verified using this method, along with group information.

References

  1. Dzuman, Z., Jonatova, P., Stranska-Zachariasova, M. et al. (2020) Development of a new LC-MS method for accurate and sensitive determination of 33 pyrrolizidine and 21 tropane alkaloids in plant-based food matrices. Anal Bioanal Chem 412, 7155–7167.
  2. European Food Safety Authority. (2011) Scientific opinion on pyrrolizidine alkaloids in food and feed. EFSA J. 9: 2406.
  3. European Food Safety Authority. (2017) Risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA J. 15: 4908.
  4. European Food Safety Authority. (2013) Scientific opinion of tropane alkaloids in food and feed. EFSA J. 11: 3386.
  5. European Food Safety Authority. (2017) Opinion of the scientific panel on contaminants in the food chain on a request from the European Commission related to pyrrolizidine alkaloids as undesirable substances in animal feed. EFSA J. 447: 1–51.
  6. European Food Safety Authority. (2018) Human acute exposure assessment to tropane alkaloids. EFSA J. 16: 5160.
  7. European Commission. Commission regulation (EC) no. 1881/ 2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union. (2006) L364, 5–24.
  8. Safety evaluation of certain food additives and contaminants: supplement 2: pyrrolizidine alkaloids, prepared by the eightieth meeting of the Joint FAO/WHO Expert Committee on Food Additives (‎‎JECFA). WHO Food additives series; 71-S2.
  9. Crews C, Berthiller B, Krska R. (2010) Update on analytical methods for toxic pyrrolizidine alkaloids. Anal Bioanal Chem. 396: 327–38.
  10. Mulder PPJ, De Nijs M, Castellari M, Hortos M, MacDonald S, Crews C, et al. (2016) Occurrence of tropane alkaloids in food. EFSA Supporting Publ. 13(12): EN-1140.
  11. Mulder PJP, Lopez Sanchez P, These A, Preiss-Weigert A, Castellari M. (2018) Occurrence of pyrrolizidine alkaloids in food. EFSA Supporting Publ. 12(8): EN-859.
  12. Highly selective analysis of pyrrolizidine alkaloids in herbal extracts by QTRAP® 6500+ and SelexION®+ Differential Mobility Separation. SCIEX technical note RUO-MKT-02-9285-A.

 

Acknowledgments

The data presented here are courtesy of and were produced in collaboration with the University of Chemistry and Technology, (Faculty of food and biochemical technology, Department of food analysis and nutrition), Prague, Czech Republic. 

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