Pyrrolizidine alkaloid profiling of four Boraginaceae species from Northern Germany and implications for the analytical scope proposed for monitoring of maximum levels

Figures & data

Figure 1. Common chemical structure of PA (comprising necine base and necic acid) and PA structures of different ester types.

Figure 2. a) Structures of common necine bases of the plant family Boraginaceae. b) Structures of typical necic acids occurring in PAs of the plant family Boraginaceae.

Table 1. PAs and PANOs of the scope of 21 PAs proposed for legislation on PAs in food and their corresponding naturally occurring isomers (given in the same row) discussed for an expansion of the scope to 35 PAs.

ester type	scope of 35 PAs
	scope of 21 PAs		respective structural isomers (14 PAs)
monoester	intermedine intermedine-NO	lycopsamine lycopsamine-NO	rinderine rinderine-NO	echinatine echinatine-NO	indicine indicine-NO
	europine europine-NO
	heliotrine heliotrine-NO
open-chain diester	echimidine echimidine-NO		heliosupine heliosupine-NO
	lasiocarpine lasiocarpine-NO
macrocyclic ester	senecionine senecionine-NO	senecivernine senecivernine-NO	integerrimine integerrimine-NO
	seneciphylline seneciphylline-NO		spartiodine spartiodine-NO
	retrorsine retrorsine-NO		usaramine usaramine-NO
	senkirkine

Table 2. Characteristic mass spectrometric fragment ions [m/z] and respective relative intensity ratios [%] used for screening and confirmation of PAs and PANOs of different necine base and ester types (Figures 1 and 2a). Base peak ions are shown in red.

1,2 unsaturated necine bases	retronecine	heliotridine	supinidine
O^9-monoester	94 (100), 96 (4), 120 (28), 138 (17), 156 (50)	94 (22), 96 (22), 120 (27), 138 (100), 156 (57)	70 (13), 94 (13), 122 (100), 140 (80)
O^9-monoester NO	94 (15), 111 (9), 138 (54), 155 (18), 172 (100)	94 (6), 111 (7), 138 (19), 155 (19), 172 (100)	70 (6), 122 (17), 140 (56), 158 (100)
open-chain diester	83 (22), 120 (100), 138 (1)	83 (2), 120 (100), 138 (4)	-
open-chain diester NO	83, 120, 136, 138*	83, 94, 120, 136, 138*	-
1,2 saturated necine bases	platynecine	-	trachelanthamidine
O^9-monoester	96 (1), 122 (18), 140 (22), 158 (100)	-	96 (5), 124 (60), 142 (100)
O^9-monoester NO	96 (1), 113 (5), 140 (1), 174 (100)	-	124 (6), 140 (4), 141 (5), 158 (100)
open-chain diester	n.d.^1	-	-
open-chain diester NO	n.d.^1	-	-

* Ion ratios and further characteristic fragment ions depend on the esterified necic acid at C7 and C9 of the necine base. ¹ Not detected.

Figure 3. Mean total PA contents of 1,2-unsaturated (green) and 1,2-saturated PAs (black) of the four Boraginaceae species. Error bars denote the scattering of the mean total PA contents in the sample size.

Table 3. Number of detected PAs and PANOs from each necine base type and their relative proportions (rel. prop.) of the mean total PA content in the four Boraginaceae species.

necine base type plant species		1,2-unsaturated^1			1,2-saturated^2
		r	h	s	t	p
Cynoglossum officinale	number of PA	-	48	-	8	1
	rel. prop. [%]	-	92	-	8	<1
Heliotropium europaeum	number of PA	-	53	4	3	-
	rel. prop. [%]	-	>99	<1	<1	-
Echium vulgare	number of PA	45	-	-	-	1
	rel. prop. [%]	>99	-	-	-	<1
Symphytum spp.	number of PA	13	-	-	-	-
	rel. prop. [%]	100	-	-	-	-

¹r = retronecine, h = heliotridine, s = supinidine. ² t = trachelanthamidine, p = platynecine

Table 4. The 10 most abundant alkaloids (sum of PAs and PANOs) of the plant species Echium vulgare. The bold alkaloids are included in the analytical scope of 21 PAs proposed for the determination of maximum levels while alkaloids given in italics are representatives of the 14 structural isomers discussed for the expansion of the scope to 35 PAs.

PA, corresponding PANO^1 and isomer	PA sum formula	detected isomers	content [mg/kg]^2	%^3	structure			no.^5
					necine base^4	necic acid^4 at
						C7 (R1)	C9 (R2)
echimidine	C20H31NO7	1	785.6	59.1	r	Ang	Ech	11
echihumiline^a or						Sen	Ech
echimidine isomer^a						Tigl	Ech
echimiplatine^a	C15H25NO6	1	256.0	19.2	r	H	Ech	5
leptanthine^a							(+)-Trach, 5ʹ-OH
vulgarine^a	C20H31NO7	-	136.8	10.3	r	H	(+)-Trach, 5ʹ-OAng	12
echivulgarine^a	C25H37NO8	-	50.7	3.8	r	Ang	(+)-Trach, 5ʹ-OH, 3ʹ-OAng	18
echiuplatine^a	C19H27NO7	-	45.6	3.4	r	Ang	3-hydroxyl-3-methylglutaric acid	9
echiumine A^a	C20H31NO6	2	12.0	0.9	r	Ang	(+)-Trach	10
echiumine B^a						iso	(+)-Trach/(-)-Vir
unknown isomer^a,b						iso	(+)-Trach/(-)-Vir
9-tigloylretronecine^a or	C13H19NO3	-	10.3	0.8	r	H	Tigl	2
9-angeloylretronecine^a or							Ang
9-senecionylretronecine^a							Sen
intermedine	C15H25NO5	1	9.2	0.7	r	H	(+)-Trach	4
lycopsamine							(-)-Vir
7-(2-methylbutyryl)-9-echimidinyl-retronecine^a,b	C20H33NO7	-	6.1	0.5	r	2-MeBut	Ech	14
uplandicine^a	C17H27NO7	-	5.6	0.4	r	Ac	Ech	7

¹PANO is presented in the form of corresponding PA, exception if no PA was detected. ² Sum of PA and PANO ³ Relative proportion of the mean total PA content. ⁴ The abbreviations of the necine base and necic acids are described in Figure 2a and b; 2-MeBut: 2-methylbutyric acid. ⁵ Internal number of PAs/PANOs used in Supplementary material (Table S1). ^a This is tentatively identified. ^b This represents a new PA/PANO for the species Echium vulgare.

Table 5. The most abundant alkaloids (sum of PAs and PANOs) and PA profile, respectively, of the plant material Symphytum spp. The bold alkaloids are included in the analytical scope of 21 PAs proposed for the determination of maximum levels while alkaloids given in italics are representatives of the 14 structural isomers discussed for the expansion of the scope to 35 PAs.

PA, corresponding PANO^1 and isomer	PA sum formula	detected isomers	content [mg/kg]^2	%^3	structure			no.^5
					necine base^4	necic acid^4 at
						C7 (R1)	C9 (R2)
intermedine	C15H25NO5	1	256.9	71.9	r	H	(+)-Trach	19
lycopsamine							(-)-Vir
PA not detected	C20H31NO7							23
echimidine NO		-	53.9	15.1	r	Ang	Ech
symlandine^a or	C20H31NO6	1	23.8	6.7	r	Ang	(-)-Vir	22
symphytine^a or						Tigl	(-)-Vir
symviridine^a						Sen	(-)-Vir
PAs not detected	C15H25NO6							20
echimiplatine NO^a,b		1	18.6	5.2	r	H	Ech
leptanthine NO^a,b							(+)-Trach, 5ʹ-OH
7-acetyllycopsamine	C17H27NO6	-	4.0	1.1	r	Ac	(-)-Vir	21

¹PANO is presented in the form of corresponding PA, exception if no PA was detected. ² Sum of PA and PANO ³ Relative proportion of the total content. ⁴ The abbreviations of the necine base and necic acids are described in Figure 2a and b. ⁵ Internal number of PA/PANO used in Supplementary material (Table S2). ^a This is tentatively identified. ^b This represents a new PANO for the genus Symphytum.

Table 6. The 10 most abundant alkaloids (sum of PAs and PANOs) of the plant species Cynoglossum officinale. The bold alkaloids are included in the analytical scope of 21 PAs proposed for the determination of maximum levels while alkaloids given in italics are representatives of the 14 structural isomers discussed for the expansion of the scope to 35 PAs.

PA, corresponding PANO^1 and isomer	PA sum formula	detected isomers	content [mg/kg]^2	%^3	structure			no.^5
					necine base^4	necic acid^4 at
						C7 (R1)	C9 (R2)
rinderine	C15H25NO5	1	15695.2	48.4	h	H	(+)-Trach	27
echinatine							(-)-Vir
heliosupine	C20H31NO7	-	13464.3	41.5	h	Ang	Ech	40
trachelanthamine	C15H27NO4	1	2457.3	7.6	t	-	(+)-Trach	25
viridiflorine^a							(-)-Vir
3ʹ-acetylheliosupine^a	C22H33NO8	-	240.0	0.7	h	Ang	Ech, 3ʹ-OAc	44
7-angeloylrinderine^a	C20H31NO6	1	189.2	0.6	h	Ang	(+)-Trach	37
7-angeloylechinatine^a							(-)-Vir
5ʹ-hydroxyrinderine^a,b	C15H25NO6	1	141.9	0.4	h	H	(+)-Trach, 5ʹ-OH	31
unknown isomer ^a,b							Ech
7-tigloylheliotridine^a or	C13H19NO3	-	121.0	0.4	h	Tigl	H	24
7-angeloylheliotridine^a						Ang
PA not detected	C15H27NO5							29
megalanthonine NO^a,b		-	48.5	0.1	p	H	(+)-Trach
3ʹ-acetylechinatine^a	C17H27NO6	-	28.4	0.1	h	H	(-)-Vir, 3ʹ-OAc	33
floridimine^a,b	C15H27NO5	1	15.3	0.05	t	-	(-)-Vir, 5ʹ-OH	28
unknown isomer^a,b							(+)-Trach, 5ʹ-OH

¹PANO is presented in the form of corresponding PA, exception if no PA was detected. ² Sum of PA and PANO ³ Relative proportion of the total content.⁴ The abbreviations of the necine bases and necic acids are described in Figure 2a and b. ⁵ Internal number of PA/PANO used in Supplementary material (Table S3). ^a This is tentatively identified. ^b This represents a new PA/PANO for the species Cynoglossum officinale.

Table 7. The 10 most abundant alkaloids (sum of PAs and PANOs) of the plant species Heliotropium europaeum. The bold alkaloids are included in the analytical scope of 21 PAs proposed for the determination of maximum levels while alkaloids given in italics are representatives of the 14 structural isomers discussed for the expansion of the scope to 35 PAs.

PA, corresponding PANO^1 and isomer	PA sum formula	detected isomers	content [mg/kg]^2	%^3	structure			no.^5
					necine base^4	necic acid^4 at
						C7 (R1)	C9 (R2)
heliotrine	C16H27NO5	1	12005.7	76.3	h	H	Heliot	57
unknown isomer^a,b
lasiocarpine	C21H33NO7	-	2263.4	14.4	h	Ang	Las	73
europine	C16H27NO6	-	879.7	5.6	h	H	Las	60
5ʹ-acetyllasiocarpine^a	C23H35NO8	1	169.4	1.1	h	Ang	Las, 5ʹ-OAc	76
isomer^a						iso	Las, 5ʹ-OAc
heliosupine	C20H31NO7	1	125.0	0.8	h	Ang	Ech	72
unknown isomer^a,b						iso	Ech
7-angeloylheliotrine^a	C21H33NO6	-	94.0	0.6	h	Ang	Heliot	71
5ʹ-acetyleuropine^a	C18H29NO7	-	63.7	0.4	h	H	Las, 5ʹ-OAc	69
3ʹ-acetylheliosupine^a	C22H33NO8	-	40.6	0.3	h	Ang	Ech, 3ʹ-OAc	75
5ʹ-hydroxyrinderine^a	C15H25NO6	-	21.8	0.1	h	H	(+)-Trach, 5ʹ-OH	58
rinderine	C15H25NO5	1	16.5	0.1	h	H	(+)-Trach	55
echinatine							(-)-Vir

¹PANO is presented in the form of corresponding PA, exception if no PA was detected. ² Sum of PA and PANO ³ Relative proportion of the total content.⁴ The abbreviations of the necine base and necic acids are described in Figure 2a and b. ⁵ Internal number of PA/PANO used in Supplementary material (Table S4). ^a This is tentatively identified. ^b This represents a new PA/PANO for the species Heliotropium europaeum.

Figure 4. Mean total PA content of the four detected PA profiles (blue) and the content of PAs covered by the scope of 21 PAs (yellow) and by the scope of 35 PAs (green). Error bars denote the scattering of the PA contents in the sample size.