Journal of Chromatography A, 1564 (2018) 1–15

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Review article

Analytical methods, occurrence and trends of tropane alkaloids and

calystegines: An update
Ana Romera-Torres, Roberto Romero-González, José Luis Martínez Vidal,
Antonia Garrido Frenich ∗
Department of Chemistry and Physics, Research Centre for Agricultural and Food Biotechnology (BITAL), University of Almería, Agrifood Campus of
International Excellence, ceiA3, Carretera de Sacramento s/n, E-04120, Almería, Spain

a r t i c l e i n f o a b s t r a c t

Article history: In the last years, the interest in secondary metabolites from plants has been growing, and even more if they
Received 6 April 2018 have or would have medical applications, as it happens with tropane alkaloids and calystegines. Therefore,
Received in revised form 1 June 2018 the number of analytical methods for the analysis of these compounds has been increasing. In this review,
Accepted 3 June 2018
the extraction methods as well as the chromatographic separation and detection techniques based on
Available online 4 June 2018
mass spectrometry to determine tropane alkaloids and calystegines in plant raw material and food have
been described. Finally, a summary of the natural occurrence of tropane alkaloids and calystegines in
Keywords:
the studied matrices, as well as their accidental presence in food, is presented, highlighting current and
Tropane alkaloids
Calystegines
future determination trends.
Analysis
© 2018 Elsevier B.V. All rights reserved.
Gas chromatography
Liquid chromatography
Mass spectrometry

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Sample preparation: extraction and clean-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Tropane alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2. Calystegines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Separation and detection techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1. Gas chromatography coupled to MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.1. Tropane alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.2. Calystegines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2. Liquid chromatography coupled to MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
3.2.1. Tropane alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2.2. Calystegines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4. Determination of TAs and calystegines in real samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Abbreviations: AA, acetic acid; ACN, acetonitrile; CE, capillary electrophoresis; CI, chemical ionization; DAD, diode array detector; DART, ambient ionization direct
analysis in real time; dSPE, dispersive solid-phase extraction; EI, electron impact; EtOH, ethanol; FA, formic acid; FID, flame ionization detector; GBC, graphitized black
carbon; HPLC, high performance liquid chromatography; HRMS, high resolution mass spectrometry; LADI, laser ablation direct analysis in real time imaging; LLE, liquid-
liquid extraction; LOD, limit of detection; LPME, liquid phase microextraction; MAE, microwave-assistance extraction; MALDI, matrix-assisted laser desorption/ionization;
MeOH, methanol; MI-SPE, molecularly imprinted solid-phase extraction; MS, mass spectrometry; NMR, nuclear magnetic resonance; NPE, nitrogen-phosphorous detector;
PLE, pressurized liquid extraction; PSA, primary secondary amine; Q, quadrupole; qOrbitrap, quadrupole-orbitrap; QqQ, triple quadrupole; qTOF, quadrupole-time of flight;
QTRAP, quadrupole-ion tramp; QuEChERS, quick, easy, cheap, effective, rugged, and safe; R, recovery; SBSE, stir bar sorptive extraction; SCX, strong cation exchange; SDME,
single drop microextraction; SFE, supercritical fluid extraction; SIM, single ion monitoring; SLE, solid-liquid extraction; SPE, solid-phase extraction; SPME, solid-phase
microextraction; TAs, tropane alkaloids; TLC, thin-layer chromatography.
∗ Corresponding author.
E-mail address: agarrido@ual.es (A. Garrido Frenich).

https://doi.org/10.1016/j.chroma.2018.06.004
0021-9673/© 2018 Elsevier B.V. All rights reserved.
2 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15

4.1. Natural occurrence of TAs and calystegines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4.2. Accidental occurrence of TAs: contaminated samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5. Conclusions and trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1. Introduction system properties. They are also useful as an antidote against poi-
soning with organic phosphorus derivatives used as insecticides
Since 1831, when K. Mein isolated atropine for the first time and also against some nerve gases [4,6–8].
(K. Mein, P.L. Geiger, K. Hesse, 1831–1833), and the elucidation of In 1988 calystegines, water soluble non-esterified alkaloids,
the structure of tropane alkaloids (TAs) by R.M. Willstätter [1], the were isolated from Calystegia sepium and their structures were elu-
interest in the secondary plant metabolites such as TAs, calyste- cidated two years later [9,10]. Although they have been extracted
gines or biogenic amines [2] has been growing. TAs are a group from several studied plant tissues, the reason why they have been
of more than 200 secondary metabolites that naturally occur in detected after 1990 is that they were discarded with the aqueous
numerous plant families, mainly in Solanaceae, Erythroxylaceae phase. While TAs are derived from tropane skeleton, calystegines
and Convolvulaceae. They have in common a tropane skeleton have a common nortropane skeleton (Fig. 1) and are characterized
(Fig. 1), characterized by a two-ringed structure with pyrrolidine by having several hydroxyl groups [11]. Calystegines A, B and C
and piperidine rings sharing a single nitrogen atom and two car- have different number of hydroxyl groups (3, 4 or 5, respectively)
bon atoms. Most of them are esters derived from organic acids (Fig. 1) [12]. Other group of calystegines, named N, possesses a
and hydroxytropanes, and many of them arise from tropine [3]. bridgehead amino group, and they are also presented with methy-
Some of the most frequent TAs are showed in Fig. 1. TAs had lated nitrogen [13]. Bearing in mind that calystegines are products
already been known in ancient time and they were used as magic from the TA pseudotropine [14], they were initially studied in
potions, recreational, shamanic and arrow poison, sedatives, hallu- Solanaceae family in the genera Atropa, Datura, Duboisia, Hyoscya-
cinogens and in folk medicine [4,5]. Nowadays, they are employed mus, and Scopolia, which are well known for containing TAs, and
in contemporary medicine such as ophthalmology, cardiology or they were found in these genera and others, such as Mandragora,
gastroenterology because their anthicolinergic (antimuscarinic), Solanum or Physalis [8]. Further researches showed their presence
antiemetic, parasympatholytic, anesthetic, and central nervous in most of the species of Convolvulaceae, Erythroxylaceae and Bras-
sicaceae families [15–17]. Calystegines are glycosidase inhibitors

Fig. 1. Structure of the main tropane alkaloids and calystegines.

 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15
Fig. 2. Some steps of the tropane alkaloid biosynthetic pathway (PMT putrescine N-
methyltransferase; TRI tropine-forming tropinone reductase; TRII pseudotropine-
forming tropinone reductase). Reproduced from Ref. [14] with permission of The
Royal Society of Chemistry.
due to their sugar-mimic structure with selective activity for cer-
tain glycosidase, and they generally compete with the substrate
for the active site. Their inhibitory activity is strong in most cases,
with potency comparable to swainsonine [14], and some of them
are ␤-glucosidase and ␣-galactosidase inhibitors [8]. Both TAs and
calystegines can be synthetized from putrescine [14], sharing sev-
eral steps of biosynthetic pathway (Fig. 2). However, from an
analytical point of view, they present several differences, and they
will be discussed separately.
Regarding TAs, several reviews have been published dis-
cussing analytical methods, highlighting the developments in gas
chromatography (GC), high-performance liquid chromatography
(HPLC), capillary electrophoresis (CE) and thin-layer chromatog-
raphy (TLC), and some of them included information on the
chemical properties, sample preparation and detection [18]. Other
reviews summarize GC-mass spectrometry (MS), HPLC-MS and
CE-MS methods for TAs determination [19,20]. Several book
chapters have been focused on TAs. For instance, extraction tech-
niques, such as solid-liquid extraction (SLE), supercritical fluid
extraction (SFE), liquid-liquid partitioning, microwave-assisted
extraction (MAE), pressurized liquid extraction (PLE), solid-phase
microextraction (SPME), among others, as well as separation and
detection techniques (GC, HPLC, CE, MS, nuclear magnetic reso-
nance (NMR)) are described [21,22], or LC–MS procedures together
with pharmacological information, physic-chemical properties,
sample preparation and chromatographic separation have been
reviewed [23].
3
Concerning calystegines, they have been studied in potato and
other food plants by GC–MS including 70 varieties of potato and
several edible fruits and vegetables, as well as potato-processed
products [24], and in potatoes, tomatoes and jimson weed with
other biologically active compounds [25]. The isolation, analysis by
TLC and GC, the occurrence in the plant kingdom as well as biosyn-
thesis, chemical synthesis and activities of calystegines have also
been described [13]. Moreover, the chromatographic techniques
used to isolate, purify, detect, and analyze polyhydroxy alkaloids,
including preparative chromatographic separation and chromato-
graphic analysis have been reviewed [12].
The aim of this review is summarizing the current analytical
methodologies used for the determination of TAs and calyste-
gines, including extraction and clean-up stages, separation by
chromatographic techniques and detection with MS, as well as
their occurrence in plant raw material and food. Bibliography was
reviewed from 2000 for calystegines and 2010 for TAs, bearing in
mind the latest reviews published [12,19].
2. Sample preparation: extraction and clean-up
During TAs and calystegines determination, sample prepara-
tion usually includes a clean-up step because the complexity of
the matrices, allowing a suitable isolation and or preconcentration
of the targeted compounds before chromatographic step, as it has
been described in the following sections.
2.1. Tropane alkaloids
TAs can be found in plant raw material as well as in food, which
have been contaminated with TAs-containing plants. According to
the revised bibliography, several extraction techniques have been
applied for the isolation of TAs from food, although SLE is mainly
used [26,27] (Table 1). However, other sample preparation tech-
niques, such as MAE [28], PLE [29], off-line-SPE [30] or online-SPE
[31], have also been used. Although most of the samples studied
are plants and contaminated food, in which this review is focused
on, TAs have also been studied in contaminated soil [32].
Because of the high polarity of these compounds, they are gen-
erally extracted using different mixtures of methanol/water. As it
can be observed in Table 1, several TAs have been isolated from
Physochlaina infundibularis using water/methanol (25/75, v/v), but
recoveries are not reported [33]. Atropine and scopolamine were
extracted from Datura innoxia, D. metel, and D. stramonium, using
a higher percentage of water, (extraction solution water/methanol
(40/60, v/v)), obtaining recoveries ranging from 95 to 101% [34].
Formic acid is usually added to the extraction solvent at ratios
from 0.2 to 2.0% to improve the extraction efficiency. A mixture
of 2% formic acid in water/methanol (93/7, v/v) was employed to
extract atropine and scopolamine from Datura innoxia, D. metel,
D. meteloides, and D. tatula, but no recoveries are reported [35]. A
lower percentage of formic acid, 1% in water/methanol (50/50, v/v),
was used to extract TAs from 18 different herbal extracts includ-
ing Hyoscyamus niger and D. stramonium [31]. Different TAs have
also been isolated from cereals and cereal-based products using
0.4% formic acid in water/methanol (40/60, v/v), with extraction
recoveries of 86–91% [36]. Using a similar extraction solvent, con-
sisting of 0.4% formic acid in water/methanol (25/75, v/v), 24 TAs
were isolated from cereal and cereal-based products with recover-
ies ranging from 59 to 133% and from tea with recoveries from
20–108% [37], which are lower than those obtained in the pre-
vious mentioned study [36]. This can be explained because the
higher number of compounds included in the new study. In addi-
tion, recoveries obtained in tea matrix are very low, which could
be caused by the complexity of the matrix. Using this last extrac-
 4
Table 1
Summary of analytes, matrixes, extraction and LC–MS techniques of TAsa .

Alkaloids Matrix Extraction and clean-up Analyzer and Column Mobile phase R LOD Ref.
time (%)

Atropine Datura species Dilution Q C18 A: 2 % FA in MeOH/ H2 O ND 0.075- [35]

Scopolamine 2 % FA in MeOH/H2 O (7/93, v/v) 15 min (7/93, v/v) 0.15 ␮g/L
B: 2 % FA in MeOH
Atropine Datura species Ultrasonication Q C18 A: 1% FA in H2 O 95- 0.050-0.1 ␮g/L [34]
Scopolamine MeOH/H2 O (60/40, v/v) 23 min B: 1% FA in MeOH 101
Atropine Datura metel Ultrasonication Q C18 A: FA in H2 O at pH 3 89- 3000 ␮g/L [39]
Scopolamine 0.1 M HCl with 70 % EtOH 50 min B: ACN 96
+ 3 TAs (50/50, v/v)
+Clean-up SPE: C18
Atropine Buckwheat Agitation QqQ C18 A: 0.2 % FA in H2 O 78- 0.03- [27]
Scopolamine 0.2 % FA and 0.2 % ACN in 20 min B: 0.2% FA in MeOH 103 0.09 ␮g/kg
H2 O/MeOH (40/60, v/v)
Atropine Datura innoxia Ultrasonication QqQ C18 A: 20 mM NH4 HCO2 and ND ND [49]
Scopolamine D. stramonium 25 % NH4 OH 26 min 0.1% FA in H2 O
Brugmansia solution + CHCl3 /MeOH (80/20, B: ACN

A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15

arborea v/v)
Atropine Buckwheat Agitation QqQ C18 A: 0.1% FA in H2 O 50- 0.04-0.2 ␮g/kg [41]
Scopolamine QuEChERS EN 10 min B: ACN 92
+ Clean-up dSPE: PSA and GBC
Atropine D. stramonium QuEChERS EN QqQ Vancomycin A: 0.1% diethanolamine in ND 0.5 ␮g/kg [42]
(-) and (+) Brugmansia +Clean-up dSPE: PSA and GBC 10 min EtOH
Hyoscyamine arborea
Contaminated
buckwheat
Atropine Scopolamine Przewalskia Agitation QqQ C18 A: 0.5% (v/v) KH2 PO4 in 82- 0.52-2.12 ␮g/L [50]
+ 2 TAs tangutica (NH4 OH solution + ACN) x3 27 min H2 O 102
+ Clean-up MI-SPE B: MeOH
Atropine Scopolamine Cereals Agitation QqQ C18 A: 6.5 mM NH4 OH in H2 O 86- 0.2-0.5 ␮g/kg [36]
+ 4 TAs Cereal-base 0.4 % FA in H2 O/MeOH (40/60, 17 min B: 6.5 mM NH4 OH in ACN/ 91
products v/v) H2 O (90/10,v/v)
Atropine Scopolamine Cereals Agitation QqQ C18 A: 10 mM (NH4 )2 CO3 in Cereal 0.05-1 ␮g/kg [37]
+ 22 TAs Pseudocereal 0.4 % FA in H2 O/MeOH (25/75, 15 min H2 O 59-
Cereal-base v/v) B: ACN 133
products + Clean-up SPE: SCX Tea
Teas 20-
108
Atropine Scopolamine Cereals Agitation QqQ Vancomycin A: 10 mM NH4 HCO2 in 61- 0.7-0.8 ␮g/kg [44]
+ 3 TAs QuEChERS EN 16 min H2 O/ACN (90/10,v/v) 111
+ Clean-up SPE: SCX B: MeOH/ACN (50/50,v/v)
Atropine Tea Agitation QTRAP C18 A: 0.1 mM oxalic acid, 80- 1-50 ␮g/kg [38]
Scopolamine Herbal Aqueous AA/MeOH (33/66, 10 min 2.0 mM NH4 CH3 CO2 and 95
v/v); NH4 OH solution pH 5-6 0.5% FA in H2 O
B: 0.1 mM oxalic acid,
2.0 mM NH4 CH3 CO2 in
MeOH/H2 O/FA (94.5/5/0.5,
v/v/v)
Atropine Scopolamine Urine Sonication QTRAP C8 A: 1 mM NH4 HCO2 and 76- 5 ␮g/L [40]
+ 2 TAs Herbal ACN/H2 O (70/30, v/v) 24 min 0.1% FA in H2 O 96
+ Clean-up LLE: Na2 CO3 and B: 1 mM NH4 HCO2 and
NaHCO3 in 0.1% FA in ACN
heptane/CH2 Cl2 /C2 H4 Cl2
Atropine Physochlaina Ultrasonication qTOF C18 A: 0.1 % FA in H2 O ND ND [33]
Scopolamine infundibularis MeOH/H2 O (75/25, v/v) 16 min B: ACN
+ 3 TAs
 Atropine Scopolamine Duboisia Ultrasonication and agitation qTOF C18 A: 0.1 % FA in H2 O ND 1.22-12.5 ␮g/L [48]
+ 4 TAs myoporoides 0.5 % H3 PO4 in H2 O 17 min B: MeOH/ACN (80/20, v/v)
D. leichhardtii
Atropine Scopolamine Cereal Agitation Orbitrap C18 A: 0.1 % FA in H2 O 60- 0.1-2 ␮g/kg [30]
+ 12 TAs Pseudocereal 0.5 % AA in H2 O/ MeOH (33/66, 18 min coupled to B: ACN 109
Cereals-based v/v) HILIC except
products + Clean-up SPE: SCX soy
Atropine Scopolamine Tea Agitation Orbitrap C18 A: 0.1 % FA in H2 O 75- ND [26]
+ 11 TAs Herbal tea 0.4 % FA in H2 O/MeOH (25/75, 18 min coupled to B: ACN 128
v/v) HILIC
+ Clean-up SPE: SCX
Atropine Animal Agitation Orbitrap C18 A: 0.1 % AA in H2 O 80- ND [68]
Scopolamine feed QuEChERS EN 14.5 min B: 0.1% AA in MeOH/ACN 120
(90/10, v/v)
Atropine Honey Agitation Orbitrap C18 A: 2 mM NH4 HCO2 , 0.5 mM ND 10-50 ␮g/kg [43]
Scopolamine Tea QuEChERS EN 29.5 min FA in H2 O
Tropine Food B: 0.5 mM FA with 2 mM

A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15

supplements NH4 HCO2 in MeOH/H2 O
(95/5, v/v)
Atropine Honey Agitation Orbitrap C8 A: 0.1% FA in H2 O 96- 0.04-0.2 ␮g/kg [46]
Scopolamine H2 SO4 + zinc dust 25 min B: MeOH/ACN (50/50, v/v) 109
QuEChERS
+ Clean-up dSPE: PSA and
Mg2 SO4
Scopolamine Herbal Sonication qOrbitrap 29.5 Biphenyl A: 0.1% FA with 5 mM ND 0.15-0.2 ␮g/L [31]
Atropine extract (18) 1 % FA in H2 O/MeOH (50/50, min NH4 CH3 CO2 in H2 O
+ 3 TAs v/v) B: 0.1% FA with 5 mM
Untarget + Clean-up online-SPE: NH4 CH3 CO2 in MeOH/ACN
hydrophilic divinylbenzene (95/5, v/v)
Atropine Dietary Agitation qOrbitrap C18 A: 0.1% FA with 5 mM 70- 10 ␮g/kg [45]
Scopolamine supplements QuEChERS 15 min NH4 HCO2 in H2 O 120
+ 2 TAs 2 % FA in H2 O (tablets and A: 0.1% FA with 5 mM
capsules) or n-hexane (softgel) NH4 HCO2 in MeOH
or nothing (liquid) + ACN
(tablets, softgel, capsules) or 2
% FA in ACN (liquid) + Mg2 SO4
and NaCl
+Clean-up dSPE: C18 and
Mg2 SO4 (softgel)
Atropine Scopolamine Datura Sonication DART-qTOF-MS ND [69]
+ 2 TAs species A mixture of ethyl acetate,
EtOH, and H2 O (33/33/33,
v/v/v)
Atropine Datura Sonication MALDI-TOF-MS ND [67]
Scopolamine seed H2 O LADI-DART-
+ 3 TAs Matrix solution: TOF-MS
2,5-dihydroxybenzoic acid in
ACN and 0.5 % trifluoroacetic
acid in H2 O (50/50, v/v)
1/10 extract/matrix
a
Abbreviations: AA: Acetic acid; ACN: acetonitrile; DART: ambient ionization direct analysis in real time; dSPE: dispersive solid-phase extraction; EtOH: ethanol; FA: formic acid; GBC: graphitized black carbon; LADI: laser
ablation direct analysis in real time imaging mass spectrometry; LLE: liquid-liquid extraction; LOD: limit of detection; MALDI: matrix-assisted laser desorption/ionization; MeOH: methanol; MI-SPE: molecularly imprinted
solid-phase extraction; ND: non defined; PSA: primary secondary amine; Q: quadrupole; qOrbitrap: quadrupole-orbitrap; QqQ: triple quadrupole; qTOF: quadrupole-time of flight; QTRAP: quadrupole-ion tramp; QuEChERS:
Quick, Easy, Cheap, Effective, Rugged, and Safe; R: recoveries; SCX: strong cation exchange; SPE: solid-phase extraction.

5
6 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15

tion solvent, 0.4% formic acid in water/methanol (25/75, v/v), 13
TAs were extracted from tea and herbal tea samples with recover-
ies between 75–128% [26], which are higher than those previously
commented [37].
Another procedure used 0.2% of acetonitrile and 0.2% formic
acid in water/methanol (40/60, v/v) to extract TAs from buckwheat.
Recoveries from 88–89% (scopolamine) and 79–111% (atropine)
were obtained without the addition of acetonitrile, whereas when
0.2% of acetonitrile was added to the extraction solvent, recover-
ies improved to 98–102% (scopolamine) and 104–105% (atropine)
[27]. Acetic acid is also added to methanol/water for the isolation
of TAs. An aqueous solution of acetic acid/methanol (33/67, v/v),
adjusted to pH 5–6 with ammonia solution, has been used to extract
atropine, scopolamine, and their N-oxides from tea and herbal tea,
achieving recoveries from 80 to 95% [38]. This extraction solution
has been used for the extraction of 14 TAs from cereals, pseudoce-
reals and cereal-base products, achieving recoveries ranging from
60 to 109%, except for some compounds in soy [30]. An extraction
using a mixture of an aqueous solution of 0.1 M hydrochloric acid,
based on the procedure of the State Pharmacopeia XI, with 70%
ethanol (50/50, v/v) has been carried out to extract atropine and
scopolamine from D. metel, obtaining recoveries from 89 to 96%
[39]. Acetonitrile/water mixtures are also employed to isolate TAs,
instead of methanol/water. Thus, a mixture of acetonitrile/water
(70/30, v/v) was used to extract the targeted compounds from
herbal samples with recoveries between 76 to 96% [40].
In the last few years, the well-known QuEChERS (Quick, Easy,
Cheap, Effective, Rugged, and Safe) extraction procedure has also
been used. TAs have been extracted from cereals and pseudo-
cereals using water and 1% formic acid in acetonitrile (v/v) [41,42]
or acetic acid instead of formic acid for the extraction of TAs from
honey, tea and food supplements [43]. Nevertheless other extrac-
tion mixtures as 0.5% formic acid in acetonitrile/water (75/25, v/v)
[44] have been used for the extraction of TAs from cereals, Datura
stramonium or Brugmansia arborea seeds. Several TAs have also
been isolated from dietary supplements, and depending on the
matrix, 2% formic acid in acetonitrile (liquid product), 2% formic
acid in water and acetonitrile (tablets and capsules products), or
n-hexane have been used as extraction solvents [45]. A QuEChERS
extraction method adding a zinc dust and 0.1 M of sulfuric acid
has been used at the beginning of the extraction of TA from honey
[46] to minimize the sample viscosity and to reduce the N-oxides-
alkaloids to alkaloids [47].
Moreover, other extraction procedures consisting of 0.5% ortho-
phosphoric acid have been applied to extract TAs from Duboisia
myoporoides and Duboisia leichhardtii, which provides a good
extraction of TAs and, in addition, decreases the content of other
compounds in the final extraction solution, like phenolic com-
pounds [48].
Additionally, TAs are extracted with basic solvents achieving
similar recoveries than those obtained when acidified extraction
solvents were utilized. For instance, atropine and scopolamine
have been extracted from Datura innoxia, D. stramonium and Brug-
mansia arborea using an ammonia solution (25%), and then a
mixture of dichloromethane/methanol (80/20, v/v) [49] has been
added to achieve an exhaustive extraction of these compounds.
On the other hand, a three-times extraction using ammonia solu-
tion and acetonitrile, followed by a SPE, utilizing a molecularly
imprinted polymer (MIP-SPE) was used to extract TAs from Prze-
walskia tangutica, obtaining recoveries from 82 to 102% [50]. This
synthetic MIP, used as sorbent, extracted selectively atropine,
scopolamine, aposcopolamine, apoatropine, and anisodamine and
provided excellent results that demonstrate the applicability of
MIS-SPE to separate and purify TAs. Moreover, atropine and scopo-
lamine were extracted using 5% potassium hydroxide in methanol
and dichloromethane from D. stramonium seeds, Fagopyrum escu-
lentum fruits, and cereal-base products, obtaining recoveries from
89 to 110% [51]. For fatty samples, fat was removed performing a
previous Soxhlet extraction, using n-hexane as extraction solvent
(Table 2).
Methanol has also been employed as extraction solvent when
MAE has been used (Table 2). For instance atropine and scopo-
lamine were extracted from Datura metel at 50–60 ◦ C for 3–12 min,
achieving recoveries from 93.5 to 109.5% [28]. This solvent has

Table 2
Summary of analytes, matrixes, extraction, and GC–MS techniques of TAs.a .

Alkaloids Matrix Extraction and Derivatization Analyzer Column R (%) LOD Ref.
clean-up and time

Atropine D. stramonium Soxhlet Hexamethyldisilazane Q DB5 MS 89-110 0.3-1 ␮g/kg [51]

Scopolamine Fagopyrum Esculentum n-Hexane x 2 80 ◦ C for 15 min 42 min Phenyl arylene
Cereal-based products 5 % KOH in polymer
MeOH + CH2 Cl2
Atropine D. metel Ultrasonication Derivatization Q HP Ultra 2 89-96 3000 ␮g/L [39]
Scopolamine 0.1 HCl with 70 % EtOH Bis(trimethylsilyl)- 50 min (5%-Phenyl)-
+ 3 TAs (50/50, v/v) trifluoroacetamide methylpoly-siloxane
+ Clean-up SPE C18 60 ◦ C for 30 min
Atropine Erythroxylum PLE – Q DB5 MS ND ND [29]
Scopolamine delagoense, MeOH 12 min Phenyl arylene
+ 6 TAs E.emarginatum 100 bar at 60 ◦ C polymer
E. pictum
67 TAs Datura stramonium L. Ultrasonic – Q HP5 MS ND ND [52]
MeOH/ACN (80/20, v/v) 70 min (5%-phenyl)-
Evaporation and methylpoly-siloxane
dissolution in 0.2 M
H2 SO4 , washed with
CHCl3 + NH4 OH
solution + CHCl3 +
Na2 SO4
Atropine Datura metel MAE 10% ((N,O- Q HP5 MS 93.5- 3000- [28]
Scopolamine Brugmansia pittieri MeOH at 50-60 ◦ C bis(trimethylsilyl) 10 min (5%-phenyl)- 109.5 3500 ␮g/kg
+ Clean-up dSPE GBC trifluoroacetamide and methylpoly-siloxane
and Z-Sep+ trimethylchlorosilane
in ACN
a
Abbreviations: ACN: acetonitrile; dSEP: dispersive solid-phase extraction; EtOH: ethanol; GBC: graphitized black carbon; LOD: limit of detection; LOQ: limit of quan-
tification; MAE: microwave-assistance extraction; MeOH: methanol; ND: non defined; PLE: pressurized liquid extraction; Q: quadrupole; R: recoveries; SPE: solid-phase
extraction.
 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15
been tested in PLE procedures to extract several TAs from differ-
ent Erythroxylum species at 100 bar and 60 ◦ C, but no recoveries
are reported [29]. In addition a mixture of methanol/acetonitrile
(80/20, v/v) has been used to extract 67 TAs from Datura stramo-
nium, followed by evaporation, dissolution in 0.2 M sulfuric acid,
and the extract was washed with chloroform, but recoveries are
not presented [52] (Table 2).
With the aim of enhance phase separation, some studies employ
extraction salts, mainly when a QuEChERS-based extraction proce-
dure is developed (Table 1). Different modifications of the original
QuEChERS have been applied, including AOAC 2007 [53] and EN
15662 method [54]. Both procedures have been applied, obtaining
similar results in relation to phase partition. For instance, mag-
nesium sulfate and sodium chloride have been used during the
extraction of TAs from dietary supplements [45], while anhydrous
sodium sulfate and ammonium acetate have been used for the
extraction of TAs from cereals and pseudo-cereals [41,42]. More-
over, magnesium sulfate and sodium acetate acidified with acetic
acid provide the AOAC buffered extraction [43]. The other variant
according to EN 15662 has been applied adding magnesium sulfate,
sodium chloride, and buffer citrate, to extract TAs from cereals [44]
and honey [46].
An additional clean-up step based on SPE has sometimes been
developed in order to remove interferences and to preconcentrate
the analytes [18], applying either off-line SPE with C18 and SCX
cartridges [26,30,37] or on-line SPE with hydrophilic divinylben-
zene cartridges [31]. In this sense, SCX cartridges were used to
preconcentrate up to 14 TAs from cereals and cereal-based prod-
ucts, obtaining recoveries from 60 to 109%, except for soy [30].
Another study also uses SCX cartridges to concentrate 24 TAs from
cereals and cereal-based products, achieving recoveries from 61 to
105% [37]. In addition, reversed phase sorbents such as C18 have
been used during off-line SPE clean-up step to concentrate atropine,
scopolamine, apoatropine, anisodamine, and aposcopolamine with
recoveries of 89% for atropine and 96% for scopolamine [39].
Dispersive solid-phase extraction (dSPE) is an alternative to
conventional SPE, and it has been used by several authors testing
different sorbents as primary secondary amine (PSA), graphitized
black carbon (GBC), Z-Sep+, or magnesium sulfate. Using PSA and
GBC as sorbents, a colorless extract and recoveries from 75 to 92%
were obtained [41]. However, PSA and magnesium sulfate provided
better recoveries and less matrix effect than SPE tested with SCX
sorbents, as well as extraction time was reduced [46]. A dSPE clean-
up with C18 and magnesium sulfate was applied to softgel dietary
supplements [45], removing nonpolar co-extracts and decreasing
the matrix effects. This clean-up seemed to be effective just in oily
matrix such as softgel, whereas GBC and Z-Sep + removed chloro-
phyll, carboxylic acids and lipids from plant matrices [28].
A liquid-liquid extraction (LLE) clean-up step has been pro-
posed for complex matrices as herbal samples. Thus, a Toxi-Tube
containing sodium carbonate and bicarbonate in a mixture of hep-
tane/dichloromethane/dichloroethane was used and it provided
better extraction recoveries than other clean-up procedures as
mixed mode cation exchange or conventional C18 SPE cartridges
[40].
2.2. Calystegines
Taking into account the high polarity and water-solubility,
calystegines are usually isolated using a mixture of water and
organic solvents (Table 3), being methanol the most widely used.
Thus, a mixture of methanol/water (50/50, v/v) was the extraction
solvent most commonly utilized, and it has been applied to extract
the calystegines A3, B2 and B4 from Solanum tuberosum [55,56],
the calystegines A3, B1, and B2 from Calystegia sepium [57], and to
evaluate the calystegines content in 45 species belonging to Ery-
7
throxylaceae family [17], in 129 species of Convolvulaceae family
[15] and in 43 species of Brassicaceae family [16].
Other mixtures with different proportions of methanol/water
have been used for the extraction of these compounds. A mixture
of methanol/water (80/20, v/v) has been applied to the extraction of
the calystegines A3 and B2 [11] and the calystegines A3, B2, B3, B4,
and N1 [58] from Solanum tuberosum. Another mixture consisting of
methanol/water (20/80, v/v) has been employed to extract several
calystegines from Mandragora autumnalis, Datura metel, Hyoscya-
mus albus, Withania somnifera, Withania frutescens, and Solanum
sodomaeum [59].
Other solvents have been used to extract calystegines from dif-
ferent matrices. For instance, ethanol/water (50/50, v/v) has been
tested to isolate these compounds from Ipomoea carnea [60], while
0.2% formic acid in acetonitrile/water (50/50, v/v) as extraction sol-
vent provided suitable results to extract the targeted analytes from
Solanum tuberosum, Solanum melongena, and Capsicum annuum
[37]. It can be noticed that the different mixtures of methanol/water
(20/80, 50/50, 80/20, v/v), ethanol/water (50/50, v/v) and formic
acid/acetonitrile/water (0.2/50/50, v/v/v) provided similar results
in relation to the number of the extracted calystegines. Moreover,
most of the studies neither discuss the selection nor the optimiza-
tion of the solvent extraction.
Once the calystegines have been extracted, and previous to the
chromatographic separation, the extracts are generally evaporated
and purified using ion exchange column or resin. For instance the
extract obtained by SLE is passed through a strong acidic cation
exchange resin; then, the resin is washed with water and finally,
the compounds are eluted with ammonium hydroxide, obtain-
ing a clearer extract [56,57]. An acidic cation exchange column
hydrogen form, washed with water and eluted with ammonium
hydroxide, followed by an anion exchange column chloride form,
eluted with water, were used to obtain a purified plant extract [59].
Other studies [11,15] only selected an acidic cation exchange resin
hydrogen form, which was washed with water, and the compounds
were eluted with ammonium hydroxide. A more complex proce-
dure of purification and isolation was performed employing acidic
cation and basic anion exchange columns, with hydrogen, amino
and hydroxyl forms [60].
3. Separation and detection techniques
Chromatographic techniques are the most powerful tools used
to separate relative small molecules and, coupled to MS, have
become the “golden standard” procedure for the determination
of TAs and calystegines in different types of matrices. Moreover,
there are scarce studies that employs other techniques, such as TLC
[61,62] and CE [63,64], or classical detectors such as flame ioniza-
tion detector (FID) [65], nitrogen-phosphorous detector (NPD) or
diode array detector (DAD) [66], which provide lower sensitivity
and selectivity than MS detectors, so MS applications have been
evaluated in this review.
3.1. Gas chromatography coupled to MS
3.1.1. Tropane alkaloids
The first determination of TAs using GC was performed in 1960
and since then, different chromatographic methods have been
developed. Due to the development of the interfaces between LC
and MS, nowadays, GC is less used than LC to analyze TAs. Although
the determination of TAs could be carried out without derivatiza-
tion, some studies perform a silylation of the targeted analytes,
so a reaction with hexamethyldisilazane at 80 ◦ C for 15 min
[51], with bis(trimethylsilyl)-trifluoroacetamide 60 ◦ C for 30 min
[39] or with 10% ((N,O-bis(trimethylsilyl) trifluoroacetamide and
Table 3

8
Summary of analytes, matrixes, extraction, and GC–MS techniques of calystegines.a .

Calystegines Matrix Extraction and purification Derivatization Analyzer Column LOD Ref.
b
A3 B2 B4 Solanum tuberosum (potato) Ultra-Turrax Derivatization Q DB5 1 mg/L [56]
MeOH/H2 O (50/50, Piyidine + hexamethyldisilazane (5%-Phenyl)-methylpoly-
v/v), /trichlorosilane (90/10, v/v) siloxane
evaporation + Al2 O3 to 50 ◦ C for 15 min
remove phenolic
compounds
Strong acidic cation
exchange resin
A3 B1 B2 Calystegia sepium Ultra-Turrax Derivatization Q HP5 – [57]
MeOH/ H2 O Pyridine + hexamethyldisilazane (5%-Phenyl)-methylpoly-
(50/50, v/v), + trimethylchlorosilane siloxane
evaporation 70 ◦ C for 30 min
Leaf tissues:
lyophilisation
and
derivatization.
Root tissues:

A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15

purified by
strong acidic
cation exchange
resin
A3 A5 B1 B2 B3 B4 C1 Mandragora autumnalis Ultra-Turrax Derivatization Q HP5 MS 1-2 mg/L [59]
N1 Datura metel MeOH/ H2 O (20/80, v/v), evaporation. Pyridine + hexamethyldisilazane (5%-Phenyl)-methylpoly-
Hyoscyamus albus Cation exchange column + anion exchange + trimethylchlorosilane siloxane
Withania somnifera Withania frutescens column (10:1)
Solanum sodomaeum 60 ◦ C for 30 min
A3 B2 Solanum tuberosum (potato) Agitation Derivatization Q SE 30 Poly(dimethyl silicone) – [11]
MeOH/ H2 O (80/20, v/v), evaporation, adjust Pyridine + N-methyl-N-trimethylsilyltrifluoro- gum phase
pH to 4.0 acetamide
Cation exchange resin 100 ◦ C for 1 h
B1 B2 B3 C1 Ipomoea carnea EtOH/ H2 O (50/50, v/v), evaporation, dissolved Derivatization Q SE-30 Poly(dimethyl silicone) – [60]
in water Pirydine + N-methyl-N- gum phase
Cation exchange column + anion exchange (trimethylsilyl)trifluoro-acetamide
column + cation exchange column 60 ◦ C for 1 h
A3 A5 A6 A7 B1 B2 B3 129 Convolvulaceae species MeOH/ H2 O (50/50, v/v), three times, Derivatization Q DB1 Dimethylpoly-siloxane – [15]
B4 C1 evaporation hexamethyldisilazane
strongly acidic cation exchange resin + trimethylchlorosilane
50 ◦ C for 15 min.
A3 A5 B1 B2 45 Erythroxylum species MeOH/ H2 O (50/50, v/v),), three times, Derivatization Q HP1 ND [17]
evaporation Piyidine + hexamethyldisilazane / trimethyl Dimethylpoly-siloxane
Strong acidic cation exchange resin chlorosilane HP5
50 ◦ C for 15 min (5%-Phenyl)-methylpoly-
siloxane
A3 A5 B2 B3 43 Brassicaceae species MeOH/ H2 O (50/50, v/v), three times, Derivatization Q HP1 Dimethylpoly-siloxane 3 mg/kg [16]
evaporation pyridine HP5
Strong acidic cation exchange resin with hexam- (5%-Phenyl)-methylpoly-
ethyldisilazane siloxane
and
trimethylchlorosilane
50 ◦ C for 30 min
A3 B2 B3 B4 N1 Solanum tuberosum Group Phureja Agitation Derivatization Q DB5 MS – [58]
Solanum tuberosum Group Tuberosum MeOH/ H2 O (80/20, v/v), evaporation, adjust N-methyl-N-trimethylsilyl-trifluoroacetamide Phenyl arylene polymer
pH to 4.0 50 ◦ C for 30 min
Cation exchange resin
a
Abbreviations: LOD: limit of detection; ND: not detected; Q: quadrupole.
b
Chemical ionization, with NH3 as reactant gas.
 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15
Fig. 3. Typical GC–MS of a mixture of silylated standard calystegines with octadecane as internal standard. Reprinted with permission from Ref. [59]. Copyright (2001)
Elsevier.
trimethylchlorosilane mixture in acetonitrile [28] have been devel-
oped. However, there are other studies that do not include this
derivatization step [29,52].
Two stationary phases have commonly been used, a phenyl ary-
lene polymer (DB5 MS) and (5%-phenyl)-methylpolysiloxane (HP
Ultra2 and HP5 MS). The length of the used columns is 30 m with
a particle size of 0.25 mm for DB5 MS and HP5 MS and 0.32 mm
for HP Ultra2. The total running time varies from 10 to 70 min
(Table 2). In relation to the analyzer, all the studies employed a sin-
gle quadrupole (Q) analyzer, with electron impact ionization (EI),
and using single ion monitoring mode (SIM).
Limits of detection (LODs) and limits of quantification (LOQs)
depend on the extraction procedure, matrix complexity and ana-
lyzer, so the reported limits are very diverse (Table 2). In addition,
depending on the study, LODs and LOQs are expressed in weight
or volume units. LODs ranged from 0.003 to 3.5 mg/kg and LOQs
ranged from 0.001 to 10.4 mg/kg. Additionally, a study presented
LODs and LOQs of 3 and 10 mg/L, respectively, which could corre-
spond to the instrumental limits [39].
3.1.2. Calystegines
Because of the low molecular weight of calystegines, high polar-
ity and high number of hydroxyl groups, they are derivatized prior
GC analysis. There are a few established procedures that employ
pyridine with silylating agents. One of the procedures was as fol-
lows: the compounds are dissolved in pyridine, and the solution
is mixed with hexamethyldisilazane and trichlorosilane (10:1, v/v),
and kept at ca. 50 ◦ C for 15 min [56]. The second one modified the
first procedure, using trimethylchlorosilane instead of trichlorosi-
lane, and keeping the reaction at 50 ◦ C for 15 min [15,17] or 30 min
at 50 [16], 60 [59] or 70 ◦ C [57]. In the last one, pyridine and N-
methyl-N-trimethylsilyltrifluoroacetamide are added to the extract
and the mixture is kept at ca. 50 ◦ C for 30 min [58], or for 60 min
at 60 ◦ C [60] or 100 ◦ C [11]. A typical GC MS chromatogram of a
mixture of silated standard calystegines is showed in Fig. 3.
Regarding to the chromatographic columns, DB5, HP5, HP5
MS fused silica capillary columns, consisting of (5%-phenyl)-
methylpolysiloxane, DB5 MS, containing a phenyl arylene polymer,
DB1 of dimethylpolysiloxane, HP1 of dimethylpolysiloxane, and
SE30 of poly(dimethyl silicone) gum phase, are the columns
employed in the separation of calystegines by GC (Table 3). Usu-
ally, the length of the column was 30 m [15–17,56,57,59], but also
9
15 m [58] and 60 m [11,60] column lengths were used. Calystegines
have usually been determined by GC-Q-MS with EI (Table 3), but
chemical ionization (CI) has also been applied achieving suitable
sensitivity [56]. The reported LOQs ranged from 3 to 6 mg/L or from
2 to 20 mg/kg, depending on the matrix.
3.2. Liquid chromatography coupled to MS
3.2.1. Tropane alkaloids
LC is the most used technique for separation of TAs. Usually,
TAs are analyzed using reversed phase mode, and the stationary
phase is non-polar (Table 1). C18 is the stationary phase most
used, although C8, vancomycin, HILIC, and biphenyl phases are
also used. C18 packed columns are principally applied to the
analysis of atropine and scopolamine [27,34,35,38,41,49]. In addi-
tion, this column has been used for the separation of several TAs
[33,36,39,48,50], highlighting the study that analyzed up to twenty
four TAs [37]. The length of the columns ranges from 50 to 250 mm
with particle sizes from 2 to 4.6 mm, being 2.1 mm the most used.
As example, a MS-chromatogram of the most abundant alkaloids in
Duboisia species, obtained with a C18 column, is showed in Fig. 4.
Two studies describe chromatographic methods using C8
columns, both of 150 mm of column length, analyzing atropine and
scopolamine [46] with 3 mm of particle size, or anisodamine, aniso-
dine, atropine, and scopolamine with 4.6 mm of particle size [40].
Generally, retention times obtained using C8 are higher than those
obtained with C18 columns.
Due to the high polarity of some TAs, a novelty approach con-
sisted of a C18 column (100 × 2.1 mm, 1.8 ␮m) coupled to a HILIC
column (100 × 2.1 mm, 3.5 ␮m), which enhanced the separation
because of the hydrophilic interactions (Fig. 5). This approach has
been applied to determine fourteen TAs in cereals and pseudo-
cereals [30] and thirteen TAs in teas and herbal teas [26]. Another
alternative is the use of a biphenyl column (150 × 3 mm, 2.7 ␮m),
which was selected for the analysis of TAs, atropine and scopo-
lamine, with other several alkaloids [31].
Atropine is a racemic mixture composed of (+)-hyoscyamine and
(-)-hyoscyamine, which is the active compound naturally occurred
and racemized to (+)-hyoscyamine during extraction process.
Their enantiomeric separation was performed using a vancomycin
bonded phase column of 150 mm of length [30]. This column
(250 × 4.6 mm) has also been used for the separation of these enan-
10 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15

Fig. 4. MS-chromatogram of the most abundant alkaloids in Duboisia spec.: scopolamine (5), norscopolamine (6), 6-␤-hydroxy-hyoscyamine (4), hyoscyamine (2),
norhyoscyamine (3), littorine (1), aposcopolamine (19). A) Standard soln. at a concentration level of 20 mg/l. B) Leaf extract of genotype C (Duboisia leichhardtii). Reprinted
with permission from Ref. [48]. Copyright (2016) Elsevier.

Fig. 5. Extracted ion chromatogram of anisodamine when different stationary
phases are used.
tiomers and other TAs, but using the conditions optimized for the
separation of the other TAs, the chromatographic separation of (+)-
hyoscyamine and (-)-hyoscyamine was not achieved and they were
quantified as sum (atropine) [44].
Regarding the aqueous phase, it is usually modified with 0.1%
acetic acid or with formic acid at concentrations from 0.1 to 2.0%
(v/v) (Table 1). Sometimes, when formic acid is used, 1–20 mM
ammonium formate or 0.2–5.0 mM ammonium acetate are also
added, as well as 0.1 mM of oxalic acid [38]. Other ammonia salts
are also added as modifiers of the aqueous phase, such as ammo-
nium hydroxide (6.5 mM) or ammonium carbonate (10 mM). When
20 mM ammonium formate and 0.1% formic acid in water are used,
better resolution, separation and peak shape than those obtained
with 0.1% formic acid in water or 20 mM ammonium formate in
water [49] were obtained. On the other hand, the use of 2 or 5 mM
ammonium formate [43,45] with formic acid in organic and aque-
ous phase avoided the formation of sodium or potassium adduct.
In addition, retention and peak shape for basic alkaloids were opti-
mized at pH 5 [49]. Other authors [37] added 10 mM ammonium
carbonate, working at pH 10, achieving suitable retention time
and separation of isobaric compounds. Moreover, 0.5% potassium
dihydrogen phosphate (v/v) is added to aqueous phase, but no
advantages have been indicated [50]. Some studies add organic sol-
vents to the aqueous phase as modifiers. For instance, methanol
[35] or acetonitrile [44] have been added to the aqueous phase at
percentages equal to or lower than 10%, improving the sensitivity
as well as the chromatographic separation of the target compounds.
Due to reversed phase columns are commonly selected, the
mobile phases consisted in methanol, acetonitrile or a mixture of
them (50/50 or 80/20, v/v) as organic phase are used, and some-
times they are acidified with formic or acetic acid (Table 1). In
addition as it happens with the aqueous phase several modifiers are
employed, such as 0.2–2.0% formic acid in methanol or oxalic acid
[38]. Similar organic phase has been prepared with 1 mM ammo-
nium formate and 0.1% formic acid in acetonitrile [40], instead of
methanol, or with 5 mM of ammonium acetate and 0.1% formic acid
 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15
in methanol/acetonitrile (95/5, v/v) [31]. Moreover, 6.5 mM ammo-
nium hydroxide in acetonitrile/water (90/10, v/v) has been used as
organic phase [36].
On the other hand, a particular study employed normal phase
for the enantiomeric separation of TAs, using as mobile phase 0.1%
diethanolamine in ethanol to achieve the separation of (+) and (-
)-hyoscyamine [42]. Although LC analysis generally takes from 10
to 30 min, with the exception of 50 min [39], the common running
time is around 15–17 min.
Regarding the analyzers, TAs have been quantified using low
resolution analyzers as Q [34,35,39], triple quadrupole (QqQ)
[27,36,37,41,42,44,49,50], or hybrid analyzer such as quadrupole-
ion trap (QTRAP) [38,40]. Moreover, high resolution mass
spectrometry analyzers, as Orbitrap [26,30,43], Q coupled to time
of flight (qTOF) [33,48] and Q coupled to Orbitrap (qOrbitrap)
[31,45,46], have also been used. The advantages of using high reso-
lution mass spectrometry (HRMS) are the high degree of selectivity
provided by the mass resolving power, allowing the unequivo-
cal identification of the targeted and untargeted compounds as
well as the profile of several plant species [31,43]. In addition, an
untargeted screening for TAs has been developed using a database
containing name, molecular formula, retention time, accurate mass
of parent ion and fragments, allowing the confirmation of aniso-
damine, tropinone and apohyoscyamine [31].
The reported LODs and LOQs are summarized in Table 1,
although some studies only report one of them. When Q ana-
lyzer is used, LODs ranged from 0.05 to 3000 ␮g/L and LOQs from
0.167 to 10,000 ␮g/L. When QqQ is used, LODs of 0.52–2.12 ␮g/L
or 0.03–1.00 ␮g/kg and LOQs from 0.1–5.0 ␮g/kg are obtained,
and QTRAP analyzer has provided LODs of 5 ␮g/L or LOQ of
1 ␮g/kg, depending on the matrix. When HRMS is applied, LODs
of 1.22–12.5 ␮g/L and LOQ of 160 ␮g/L were obtained when
qTOF analyzer is used, while with Orbitrap, LODs ranged from
0.04–50 ␮g/kg and LOQs from 0.1–50 ␮/kg. Finally, if qOrbitrap ana-
lyzer is used, LODs from 0.15–0.20 ␮g/L or 10 ␮g/kg and LOQs from
0.51–0.65 ␮g/L or 50 ␮g/kg are achieved. In relation to sensitivity,
QqQ analyzer shows excellent LODs and LOQs. Nevertheless, qOr-
bitrap improves selectivity, providing a more reliable identification
of the compounds because of the accurate mass.
3.2.2. Calystegines
Up to now, calystegines have been widely analyzed by GC, but
there are few reports focused on LC analysis. One reason is because
they are hydrophilic, and they are not amenable to be separated
by conventional LC reversed phase columns. Moreover, the chro-
matographic separation of isomeric compounds is a challenge, and
usually a chiral column is needed. The two published studies used
an aqueous phase with ammonium acetate, at 5 and 20 mM, but
the organic phases are pure methanol and acetonitrile, respectively.
The first one is a collaborative study and employed a vancomycin
chiral column (150 × 2.1 mm, 5 ␮m) coupled to both QqQ and qOr-
bitrap for the analysis of the calystegines A3, A5, B1, B2, B3, and B4,
reaching LODs from 0.25–1.0 mg/kg and LOQs from 1.0–2.5 mg/kg,
with recoveries ranging from 83 to 100% [37]. When the chiral
column was used, the calystegines are completely separated, and
eluted with retention times from 2.4 (B2) to 6.7 min (A3). In the sec-
ond one, a HILIC column (100 × 3 mm, 3 ␮m) coupled to a QTRAP
analyzer was used to analyze the calystegines A3, B2, and B4,
achieving LODs of 0.4–0.6 mg/kg [55]. The calystegines were also
entirely separated, with retention times of 4.1 (B2), 4.5 (B4) and
5.1 (A3) min.
4. Determination of TAs and calystegines in real samples
Two sample groups can be distinguished. The first one corre-
sponds to the raw material in which TAs and/or calystegines are
11
naturally present, while the second one is composed by food that
has been contaminated with plants or part of plants-containing TAs.
Up to now, the study of calystegines has been focused on the first
group, while the presence of TAs has been examined in both groups
due to the toxicity of some of these compounds (Tables 4 and 5).
4.1. Natural occurrence of TAs and calystegines
Regarding the first group, TAs are generally analyzed in sev-
eral Datura, but also in Brugmansia and Hyoscyamus species, being
atropine and scopolamine the most studied compounds (Table 4).
Atropine and scopolamine have been determined at concentra-
tions ranging from 378 to 1283 mg/kg, respectively [51] in Datura
stramonium seeds, while their concentration varied from 65–2788
(atropine) and 448–2020 mg/kg (scopolamine) in D. metel and Brug-
mansia pittieri [28]. In other studies, the concentrations ranged
from 20–5910 ␮g/kg of atropine and 10–4530 ␮g/kg of scopo-
lamine in D. innoxia, D. metel and D. stramonium [34], or from
0.19 to 31.00 mg/L of atropine and 11.00–400.04 mg/L of scopo-
lamine in D. innoxia, D. metel, D. meteloides, and D. tatula [35].
In order to increase the information related to the presence of
TAs, other studies analyzed several TAs, as atropine, scopolamine,
apoatropine, aposcopolamine, and anisodamine, in D. metel but
only concentrations of atropine (10–900 mg/kg) and scopolamine
(200–1700 mg/kg) were reported [39]. These concentrations are
much higher than those obtained in 18 herbal extracts, where
atropine, scopolamine, apoatropine, anisodamine, and tropinone
were analyzed, presenting concentration ranging from 0.18 (scopo-
lamine) to 204 mg/kg (atropine) [31]. In addition, a study, focused
on the separation and determination of (−) and (+)-hyoscyamine
in D. stramonium and Brugmansia arborea seeds, obtained concen-
trations ranging from 16.21 ((-)-hyoscyamine) to 1454.30 mg/kg
((-)-hyoscyamine) [42].
Most of the studies analyzed calystegines in Solanum tubero-
sum (potato), but they were also determined in Capsicum annuum,
Calystegia sepium or Ipomoea carnea, among others (Table 5). In
Solanum tuberosum tubers, the calystegines A3 and B2 were deter-
mined at concentrations from 0.2 (A3) to 2100.0 (B2) mg/kg [11]. In
another study the total content of calystegines, including A3, B2 and
B4, has been determined in tuber and plants during different stages
and after harvest, detecting concentrations up to 3000 mg/kg [56].
It was noticed a clear variation of the calystegines content after
harvesting, observing the highest concentration after 5 months of
cold storage, but 8 months after harvesting, this concentration was
reduced up to 50%. Additionally, these calystegines were deter-
mined in potato tubers under wounding and light exposure [55],
and concentrations from 35 (A3) to 84 (A3) mg/kg were found. Sev-
eral calystegines were analyzed in two Solanum tuberosum groups,
Phureja and Tuberosum, finding concentrations from 7.4 (N1) to
10,145.0 (B2) mg/kg and different concentrations can be observed
within the several parts of the plant as it is shown in Fig. 6 [58].
Other studies analyzed up to 404 samples consisting of Solanum
tuberosum, Solanum melongena (eggplant) and Capsicum annuum
(bell pepper) and concentrations ranged from 0.3 (B4) to 409.9 (A3)
mg/kg [37] were obtained. The accumulation of calystegines has
been studied in Calystegia sepium during the growth of the plant.
The calystegine A3 was detected at the highest concentration and
the maximal accumulation of total calystegines was in the late
culture stage at 1500 mg/kg [57], while in Ipomoea carnea, the con-
centrations of individual calystegines ranged from 40 (B3) to 210
(B2) mg/kg [60]. An investigation of the content of calystegines in
the Solanaceae family, including Datura metel, Atropa belladonna,
Hyoscyamus albus, and Brunfelsia nitida among others, revealed the
occurrence of the calystegines A3, A5, B1, B2, B3, B4, C1, and N1 [59].
The calystegines content presents several variations within the
species. Datura metel does not present any of the studied calyste-
12 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15

Table 4
Summary of analyzed TAs and concentrations found in real samples.

Analytes Matrix Concentration range Observations Ref.

Atropine Scopolamine Datura metel Atropine 64.67-2788 mg/kg [28]

Brugmansia pittieri Scopolamine 448.2-2020 mg/kg
Atropine Datura species Atropine 0.19-37.00 mg/L [35]
Scopolamine Scopolamine 11.00-400.04 mg/L
Atropine Datura species Atropine 20-5910 mg/kg [34]
Scopolamine Scopolamine 010-4530 mg/kg
Atropine Datura stramonium seed Atropine 1283 mg/kg [51]
Scopolamine Scopolamine 678 mg/kg
Atropine Buckwheat Atropine 1.1-111 mg/kg 16 food samples, [51]
Scopolamine Buckwheat-deribed products Scopolamine 0.07-66 mg/kg positive in all
Contaminated buckwheat contaminated
Atropine D. stramonium seeds (-)-Hyoscyamine [42]
(-)-Hyoscyamine Brugmansia arborea seeds 326.91-1454.30 mg/kg
(+)-Hyoscyamine (+)-Hyoscyamine 16.21-17.89 mg/kg
Atropine Contaminated buckwheat (-)-Hyoscyamine 0.10-0.17 mg/kg [42]
(-)-Hyoscyamine (+)-Hyoscyamine 0.01-0.01 mg/kg
(+)-Hyoscyamine
Atropine Buckwheat Atropine 13.9-83.9 ␮g/kg 26 samples, 11 % [27]
Scopolamine Scopolamine 5.7-10.4 ␮g/kg positives
Atropine Buckwheat <LOD 8 samples [41]
Scopolamine
Atropine Animal Atropine 7.2 ␮g/kg 32 samples [68]
Scopolamine feed
Atropine Honey Atropine up to 3.8 ␮g/kg 40 samples, 22% [46]
Scopolamine samples with atropine
Atropine Honey <LOD Screening method [43]
Scopolamine Tea
Tropine Food
supplements
4 TAs Urine Non defined 269 samples, 10 % [40]
Herbal positives
4 TAs Dietary supplements <LOD 23 samples [45]
5 TAs Herbal 0.18 (scopolamine)-204 mg/kg 18 samples [31]
Untarget extract (18) atropine
5 TAs Datura metel Atropine 10-900 mg/kg [39]
Scopolamine 200-1700 mg/kg
5 Tas Cereal 2.5-12.7 ␮g/kg (atropine) [44]
Pseudo cereal
6 TAs Cereal-based products 0.42 (scopolamine)-65.6 (atropine) 113 samples, 22 % [36]
␮g/kg positive
13 TAs Tea 5 (apoatropine)-4340 ␮g/kg (sum of 11 samples, [26]
Herbal tea physoperuvine, pseudotropine and 55 % positive
tropine)
14 TAs Cereal, pseudo cereal 3 (littorine)- 23 ␮g/kg (scopolamine) 15 samples, [30]
Cereal-based products 13 % positive
24 TAs Cereal, pseudo cereal 0.05 (atropine, scopolamine) - 1998.0 1709 samples [37]
Cereal-based productss (tropine) ␮g/kg
Tea

Table 5
Summary of analyzed calystegines and concentrations found in samples.

Analytes Matrix Concentration range Ref.

A3 B2 Solanum tuberosum 0.2 (A3)-2100 (B2) mg/kg [11]

A3 B2 B4 Solanum tuberosum Up to 3000 (B2) mg/kg [56]
A3 B2 B4 Solanum tuberosum 23– 84 (A3) mg/kg [55]
A3 B1 B2 Calystegia sepium Up to 1500 mg/kg [57]
B1 B2 B3 C1 Ipomoea carnea 4 (B3)-21 (B2) mg/kg [60]
A3 A5 B1 B2 B3 B4 Mandragora autumnalis Up to 357 (C1) mg/kg [59]
C1 N1 Datura metel
Hyoscyamus albus
Withania somnifera
Withania frutescens
Solanum sodomaeum.
A3 A5 B1 B2 45 Erythroxylum species Up to 2500 (B2) mg/kg [17]
46 samples
A3 A5 B2 B3 43 Brassicaceae species Up to 5000 mg/kg [16]
A3 A5 A6 A7 B1 B2 129 Convolvulaceae species Non defined [15]
B3 B4 C1
A3 B2 B3 B4 N1 Solanum tuberosum 7.4 (N1)-10,145 (B2) mg/kg [58]
Group Phureja Solanum tuberosum
Group Tuberosum
A3 A5 B1 B2 B3 B4 Solanum tuberosum Solanum melongena Capsicum annuum 0.3 (B4)-409.9 (A3) mg/kg [37]
404 samples
 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15
Fig. 6. Total ion chromatogram (TIC) traces showing the distribution of calystegines
in extracts of (A, B) tuber flesh, (C) peel, and (D) sprouts of Solanum phureja line DB
333-16. (1) Primary internal standard tropine (TMS); calystegines: (2) A3 (TMS)3 ,
(2a) B4 (TMS)4 , (2b) B3 (TMS)4 , (3) A3 (TMS)4 , (3a) X2 (TMS)2 , (3b) N1 (TMS)3 , (4) B2
(TMS)4 , (5) B2 (TMS)5 ; (6) secondary internal standard perseitol (TMS)6 . Reprinted
with permission from Ref. [58]. Copyright (2008) American Chemical Society.
gines, while Brunfelsia nitida presented the highest concentration
at 357 mg/kg of calystegine C1. When calystegines were studied in
46 samples of 45 Erythroxylum species, the calystegines A3, A5, B1,
and B2 were found, and the maximum concentration achieved was
2500 mg/kg corresponding to the calystegine B2 [17], whereas in
43 Brassicaceae species, the calystegines A3, A5, B2 and B3 were
determined at concentrations up to 5000 mg/kg [16] and in 129
Convolvulaceae species, the calystegines A3, A5, A6, A7, B1, B2,
B3, B4, and C1 were studied, but no concentrations are reported
[15], showing that tetrahydroxylated alkaloids B2 and B1 are the
most frequent compounds (90% and 68% of the positive species,
respectively) followed by the trihydroxynortropane A3 (38%).
4.2. Accidental occurrence of TAs: contaminated samples
Concerning contaminated samples by TAs, atropine and scopo-
lamine are the most frequently determined and, although cereals
and pseudo-cereals are the most studied matrices, there are
also a number of studies focused on herbs, dietary supplements
13
and honeys (Table 4). For instance, 26 samples of buckwheat
were investigated, and atropine and scopolamine were found in
11% of them at concentrations ranging from 13.9–83.9 ␮g/kg and
5.7–10.4 ␮g/kg, respectively [27]. However, in another study, 8
samples of buckwheat were analyzed, but neither atropine nor
scopolamine were determined above the LODs [41]. Similar results
were found [51] in buckwheat and buckwheat-derived products,
but atropine and scopolamine were only detected in contami-
nated buckwheat with Datura stramonium at concentrations from
1.1–111.0 mg/kg of atropine and from 0.07–66.00 mg/kg of scopo-
lamine. Moreover, multianalyte studies have been developed in
cereal and pseudo cereals-based products. One study determined
five TAs in several cereals, but only atropine and scopolamine
were detected at concentrations ranging from 2.5 to 12.7 ␮g/kg of
atropine [44]. In another study, 6 TAs were analyzed in 113 cereal
and pseudo-cereal samples, finding the lower concentration of
scopolamine (0.42 ␮g/kg) and the higher of atropine (65.60 ␮g/kg)
in 22% of the samples [36], whereas in another research 14 TAs
were determined in 15 cereal and pseudo-cereal samples, detect-
ing up to 5 TAs at concentrations from 3 (littorine) to 23 ␮g/kg
(scopolamine), and 13% of samples contain TAs [30]. A collaborative
work [37] monitored the presence of 24 TAs in 1709 samples com-
prising cereal, cereal-based products, and tea. The results showed
that some TAs were detected in 21% of flours, 20% of cereal-based
products or 70% of teas, finding concentrations ranging from 0.05
(atropine, scopolamine) to 1998.00 ␮g/kg (tropine) [37]. In another
work [26] 13 TAs were investigated in 11 samples of teas and herbal
teas, and concentrations from 5 (apoatropine) to 4340 ␮g/kg (sum
of physoperuvine, pseudotropine and tropine) were obtained, with
55% of positive samples.
On the other hand, 40 honey samples were analyzed (22% posi-
tive samples) and concentrations up to 3.8 ␮g/kg of atropine were
found [46], while a screening study of atropine, scopolamine, and
tropine performed in honey, tea and food supplements did not
reveal any of the targeted compounds above their LODs [43]. Neg-
ative results were also obtained when 4 TAs were determined in
dietary supplements [45]. Nevertheless, during the screening of
urine and herb samples, it was observed that TAs were detected
in 18 out of 269 of studied samples [40].
Moreover, there are several reports focused on different pur-
poses than the quantification of TAs and calystegines. For example,
a screening method of plant toxins using GC–MS has been applied
for the monitoring of seasonal alteration in concentrations of TAs
as precursors of cocaine in South African Erythroxylum species [29].
Another screening method, focused on the identification of up to 67
TAs in Datura stramonium revealed the occurrence of nine new TAs
for the first time in this plant [52]. In addition, a screening method,
which did not use chromatographic separation, has been developed
to obtain the spatial distribution in real time of TAs without matrix
pretreatment. The method consists of a laser ablation-direct analy-
sis in real time imaging mass spectrometry (LADI-MS) and it can be
applied to determine natural products of a wide range of polarities
in a diversity of samples types [67].
5. Conclusions and trends
There is no doubt that there is a growing interest in secondary
metabolites of plants such as ATs and calystegines. That is why
in the last few years a large number of analytical methods have
been published for their determination, although most of them use
similar determination techniques.
Thus, SLE is still the extraction technique most widely used
because its simplicity and suitable results, but other alternatives
as PLE or MAE have also been tested. Because the complexity of
the matrices where these compounds are found, a clean-up step is
14 A. Romera-Torres et al. / J. Chromatogr. A 1564 (2018) 1–15
commonly needed, and SPE and dSPE have been mainly used. Thus,
during calystegines determination, a purification step based on ion
exchange columns or resins are commonly needed. Nevertheless, in
the next few years, more efforts should be focused on the applica-
tion of micro-extraction techniques, as solid phase microextraction
(SPME), liquid phase microextraction (LPME), stir bar sorptive
extraction (SBSE), or single drop microextraction (SDME), in order
to minimize solvent, pre-treatment stage and time, taking into
account the main principles of green chemistry.
Chromatographic techniques coupled to Q or QqQ have been
used for the determination of TAs and calystegines, being TAs ana-
lysed by LC and calystegines by GC, bearing in mind the different
physic-chemical properties of both classes of compounds. Consid-
ering that calystegines have to be derivatized for GC determination,
more methods based on LC are demanding in order to avoid this
step, simplifying sample treatment.
Furthermore, the scope of these analytical methods could be
increase if high resolution analyzers (qTOF, Orbitrap, qOrbitrap)
are also being applied. These analyzers provide suitable sensitiv-
ity and better specificity than QqQ for the analysis of secondary
metabolites and allow the identification of targeted and untargeted
compounds, so unknown TAs or calystegines could be detected
in cereals, pseudocereals, potato, tomato, and cereals-based prod-
ucts, which are the most contaminated matrices by TAs. In addition
to atropine and scopolamine, which are the most studied TAs or
calystegines A3 and B2, which are the most frequently detected in
potato, other compounds can also be detected using these analyz-
ers. They also allow the use of fingerprinting approaches, providing
a suitable tool to differentiate between species (i. e. wild and
hybrids species) or for quality assessments.
Therefore, full scan-HRMS is a powerful tool for the analysis of
targeted and untargeted compounds, allowing the development
of unknown screening in which metabolites or untargeted com-
pounds could be tentatively identified. In addition, metabolomic
approaches can be developed. In this way untargeted alkaloids can
be tentatively identified on the basis of exact mass and isotopic
pattern, highlighting the enormous potential of this technique on
metabolomic research, based on generic extraction approaches that
allows suitable extraction of a wide scope of compounds.
Acknowledgments
The authors gratefully acknowledge to the Spanish Ministry of
Economy and Competitiveness (MINECO) and FEDER (project ref.
CTQ2015-69899-R) for financial support.
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