Because of their hepatotoxic and carcinogenic effects, authorities set limits for plants preparations that may contain pyrrolizidine alkaloids (PA) such asBorago officinalis L. According to the « Plants decree » (2014, June 24th) they should not contain more than 1 ppb of PA. However, numerous weeds likeEchium vulgare L. or Senecio vulgaris L. synthesise this type of alkaloids, and cross contaminations that may occur during harvests are frequent andinevitable. This is why health authorities, notably Swissmedic, strongly advise to extend the PA quantification assay to all the plants growing in the samebiotope as these weeds.Considering the phytosanitary issues that may induce PA ingestion and the hundreds of PA referenced in the literature, it seems paradoxical to evaluate thePA content of commercial preparations according to a short list of commercially available analytical standards that would certainly lead to false negatives.These are the reasons why Botanicert wanted to develop an extensive method for the dosage of PA by HRMS.

NEWSLETTER 19 – DOSAGE OF PYRROLIZIDINE ALKALOIDS

INTRODUCTION

Method development for the dosage of total PA

Our study is based on the fact that PA couldexist under two forms: saturated orunsaturated. Each form can be of 3 classes(monoester, diester and cyclic diester) and eachclass may be present under 3 different types (N-base, N-oxide and otonecine). It results in 18configurations to which correspond 18 familieswith particular fragmentation. The analyticalprocess involves the screening of a large PAmass range in HRMS. The second step is thestudy of the fragmentation of suspected PA toidentify their family. To be as relevant aspossible, the PA content will be expressed as aPA belonging to the same family andcommercially available as an analytical standard.

BOTANICERT, ANALYTICAL LABORATORY EXPERT IN THE PLANT FIELD, contact@botanicert.com – 04.89.85.60.27

SEPTEMBER 2017

Customer samples analysis

2

STUDY

CONCLUSION

1

How to guarantee the safety of plants based preparations that may contain pyrrolizidine alkaloids ?Thanks to its extensive PA study and LOD of 1 ppb, the UHPLC/HRMS method dedicated to PA identification and quantification developed by Botanicert meetsthe regulatory requirements.However, contaminations are frequent and inevitable. This is why, PA monitoring may be completed by a toxicity threshold study in order to justify the safety ofplants based preparations.

Different PA families

Unsaturated pyrrolizidine

Saturated pyrrolizidine

cyclic diester

monoester

N-base

N-oxide

otonecine

Customer sample: TIC CV 30V

B

A Mass spectra of A

HRMS formula: C18H23NO5

Typical PA fragments Unsaturated cyclic diester N-base

Mass spectra of B

HRMS formula: C18H23NO5

Typical PA fragments Unsaturated cyclic diester N-oxide

KNOWNsenecyphilline

(analytical standard available)

UNKNOWNexpressed as retrorsine

(available analytical standard from unsaturated cyclic diester

cyclic N-oxide family)Δ28 amu

Δ28 amu

Customer samples: molecules A and B have close and odd masses

- Mass spectrum of A: the combination of these fragments is specific to the unsaturated cyclic diester N-base PA family. In addition, the molecular formulasuggested by HRMS corresponds to a known molecule : seneciphylline (analytical standard available). A could be identified and quantified asseneciphylline.

- Mass spectrum of B: the combination of these fragments is specific to the unsaturated cyclic diester N-oxide PA family, but the molecular formulasuggested by HRMS doesn’t correspond to a commercialised analytical standard. However, retrorsine (analytical standard available) belongs to the samefamily. This unkown PA could be quantified and expressed as retrorsine.

diester