LC-MS Quantitative Determination of Atropine and ... - Semantic Scholar

LC-MS Quantitative Determination of Atropine and Scopolamine in the Floral Nectar of Datura Species

´ gnes Farkas2, Silvia Jakabova´1, Ivett Bacskay1, Ferenc Kila´r1,3, Attila Felinger1,& Borba´la Boros1, A 1 2 3

Department of Analytical and Environmental Chemistry, University of Pećs, Ifju´sa´g u´tja 6, 7624 Pećs, Hungary; E-Mail: [email protected] Institute of Pharmacognosy, University of Pećs, Ro´kus utca 2, 7624 Pećs, Hungary Institute of Bioanalysis, University of Pećs, Szigeti u´t 12, 7624 Pećs, Hungary

Received: 15 October 2009 / Revised: 31 January 2010 / Accepted: 7 February 2010

Introduction

Abstract The present study aimed at determining selected alkaloid components in the nectar of Datura species, to elucidate whether the alkaloid content of the floral nectar can lead to intoxication. A simple and rapid liquid chromatography coupled with electrospray mass spectrometry analysis was developed for the quantitative determination of atropine and scopolamine, the main toxic alkaloids of the Datura species. This method allowed the direct coupling of an electrospray mass selective detector to the LC system. Under these conditions, atropine and scopolamine were well separated from other components and detected with mass spectrometry (mass selective detector). Simultaneous determination of atropine and scopolamine was performed with gradient elution on an Ascentis Express C18 (Supelco) reversed-phase column based on a new fused core particle design. Liquid chromatography coupled with electrospray mass spectrometry was used in positive ion mode. Atropine and scopolamine produced protonated species at m/z 290 and 304 (which are also the base peaks). Our data confirmed that the alkaloid characteristics for the vegetative and reproductive parts of the Datura plants may also occur in the nectar secreted by the flowers. In Datura species with large flowers and high nectar amounts, the alkaloid content increases proportionately and thus the nectar may be a potential source of intoxication.

Keywords Column liquid chromatography-mass spectrometry Electrospray MS Atropine and scopolamine Datura floral nectar

Presented at: 8th Balaton Symposium on High-Performance Separation Methods, Sio´fok, Hungary, September 2–4, 2009.

The tropane alkaloids atropine and scopolamine are found in several members of the Solanaceae family, the most common sources being Atropa belladonna and representatives of the genera Datura and Duboisia. Datura (thorn apple or angel’s trumpet) species are indigenous to America and Asia [1], but are widespread as ornamentals in Europe, as well. Certain thorn apple species, such as D. innoxia and D. metel are cultivated also for medicinal purposes, being good sources of the alkaloid scopolamine [1]. On the other hand, Datura species are known as hallucinogenic plants that may cause serious poisoning [2–5]. The main toxic principles are the tropane alkaloids hyoscyamine, which form a diastereomeric (epimeric) mixture known as atropine (upon isolation) and scopolamine [6]. Atropine and scopolamine have anticholinergic properties and have legitimate medical applications in very low doses. Atropine (see Fig. 1) has a molecular mass of 289.37. Generally, atropine causes blurred vision, suppressed salivation, vasodilation, increased heart rate, and delirium [6–11]. It also reduces rigidity in parkinsonism and is used as an antidote to poisoning with parasympathomimetic agents, e.g.

Original DOI: 10.1365/s10337-010-1524-y Ó 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH

Table 1. LOD and LOQ results for atropine and scopolamine

CH 3 N

O

OH

Sample

LOD (pg mL-1)

LOQ (pg mL-1)

Atropine Scopolamine

75.08 150.15

250 500

Table 2. Intraday precision of the retention time of atropine and scopolamine in standard solution

O Fig. 1. Structure of atropine

Standard solution (500 ng mL-1 for each alkaloid)

Scopolamine

Atropine

Retention time (min)

RSD%

Retention time (min)

RSD%

4.056

0.94

5.414

0.76

CH3 N O

Data are means from six determinations each (± standard deviation) Table 3. Interday precision of the retention time of atropine and scopolamine in standard

O

OH O

Fig. 2. Structure of scopolamine

nerve gases and organophosphorus insecticides [11]. Scopolamine (also called hyoscine, Fig. 2) has a molecular mass of 303.36. Scopolamine is an antimuscarinic agent (used as an analgesic) and a smooth muscle relaxant. It is also an antispasmodic agent with antinauseant properties, and is extensively used in the treatment of motion sickness and in pre-operative medication [7, 8, 11]. The two anticholinergic alkaloids may be present in animal feed and grain, e.g. in the case of contamination with D. stramonium seeds, therefore developing accurate analytical methods for the analysis of these food types is of high importance [12]. Alkaloid content and composition in various Datura species may vary depending on the species, the plant part [13, 14], the phenological stage [15] and also the place of growing [16]. The roots and aerial parts of D. innoxia contain the main alkaloid scopolamine but hyoscyamine is also present in significant amounts [17]. Besides these two alkaloids, flowers contain tyramine, while meteloidine can be detected in the stems. The seeds are characterized by the dominance of hyoscyamine (0.21%) over scopolamine (0.09%) [1]. The alkaloid content of D. metel varies to a great extent depending on the plant part concerned: roots 0.1–0.2%,

solution Standard solution (500 ng mL-1 for each alkaloid)

Retention time of scopolamine (min)

Retention time of atropine (min)

1 Day* 2 Day* 3 Day* Retention time (min) RSD%

4.056 4.067 4.059 4.061 0.79

5.414 5.445 5.432 5.430 0.68

* Data are means from six determinations each/day (± standard deviation)

leaves 0.5%, flowers 0.1–0.8%, fruits 0.12% and seeds 0.2–0.5% alkaloid [18]. D. metel was reported to contain the highest amount of scopolamine among thorn apple species. The analysis of tropane alkaloids in the seeds, flowers, and leaves of downy thorn-apple showed that scopolamine was always dominant over hyoscyamine [15, 19]. The root was the organ which often accumulated higher amounts of atropine [15]. Other alkaloids include atropine, meteloidine, norscopolamine, norhyoscyamine and datumetin [20]. The alkaloid profile is helpful for the classification of the family Solanaceae, to which Datura species belong [21, 22]. Consumption of any part (e.g. flower, leaves or seeds) of thorn apples, either intentionally or accidentally, may lead to intoxication [2–5]. Various insect pollinators have also been reported to become addicted to the narcotic nectar of Datura flowers [23], but to date little is known about the presence of toxic alkaloids in the secretory products of the plants. No data are available concerning the actual alkaloid content of nectar, the aqueous sugar solution produced by the flowers,

which, together with pollen, serves as the most important food and energy source for pollinating insects. The analysis of tropane alkaloids has been studied using different instrumental techniques, including: atomic emission spectrometry (AES) [24], atomic absorption spectrometry (AAS) [25], UV spectroscopy [12], enzyme-linked immunosorbent assay (ELISA) [12], gas chromatography (GC) [26], gas chromatography-mass spectrometry (GCMS) [12, 21, 27], quantitative thin-layer chromatography (TLC) [12, 26, 28], high performance thin-layer chromatography–densitometry (HPTLC–densitometry) [29], capillary electrophoresis (CE) [26, 30–32], micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) [33], and more recently, reversed-phase liquid chromatography with UV detection (RP-LC) [12, 21, 26, 29, 34–38]. Several procedures employing liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS-MS) have been developed for the simultaneous determination of atropine and scopolamine in biological fluids [39–44]. Usually these samples were Original

extracted and cleaned-up by liquid–liquid extraction (LLE) [40, 44], and by solid-phase extraction (SPE) using conventional sorbents (on C18 cartridges) [39–41, 43]. LC is considered as a reliable and sensitive method for identification and confirmation of atropine and scopolamine in biological samples [43]. The limit of detection (LOD) for that method equipped with PDA detectors is usually less than 1 lg mL-1 for atropine as well as for scopolamine [29, 43]. LC-MS is more sensitive and it can achieve LOD of 10 pg mL-1 for atropine and 100 pg mL-1 for scopolamine [43]. Chen et al. [39] achieved 5 ng mL-1 LOD for atropine using LCMS-MS. Several studies confirmed that LC-MS as technique provides an effective tool for quantification of tropane alkaloids [32, 39, 43]. The present study aimed at determining alkaloid components in the nectar of various Datura species, and to elucidate if the alkaloid content of the floral nectar can lead to intoxication.

Experimental Chemicals and Reagents (±)-Atropine (99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). (-)-Scopolamine hydrochloride (Reag. Ph. Eur.) was purchased from Merck (Darmstadt, Germany). Formic acid (eluent additive for LC-MS) and water (LC-MS Chromasolv) were obtained from Fluka (Buchs, Switzerland). Methanol (LC-MS Chromasolv) was purchased from Riedel-de Hae¨n GmbH & Co. (Seelze, Germany).

Samples Plant Samples

The seeds of the following species were obtained from various botanical gardens, as listed below—indicating also the year of sample collection: Datura innoxia Mill. (CH): Botanical Garden, Zurich University, Switzerland – 2007 Original

Table 4. Intraday precision of the concentration of atropine and scopolamine in Datura nectar samples Alkaloid

Atropine Scopolamine

Datura innoxia (13 July 2009)

Datura metel L. (HU)

Concentration (lg mL-1)

RSD%


RSD%

5.70 157.20

4.04 1.97

4.11 128.22

3.41 1.31

Data are means from four determinations each (± standard deviation)

Table 5. Interday precision of the concentration of atropine and scopolamine in Datura nectar samples Alkaloid

Datura innoxia (13 July 2009) Concentration (lg mL-1)

Datura metel L. (HU) Concentration (lg mL-1)

1 Day* Atropine Scopolamine

5.70 157.20

4.11 128.22


6.25 151.18

3.85 135.72


5.92 163.08

4.78 131.05

Concentration (lg mL-1) Atropine 5.96 Scopolamine 157.15

4.25 131.66

RSD% Atropine Scopolamine

3.36 1.97

4.47 1.57

* Data are means from four determinations each

D. innoxia Mill. (PL/1): Hortus Plantarum Medicinae, Poland – 2007 D. innoxia Mill. (PL/2): Department of Medicinal Plants, Poznan, Poland – 2007 D. innoxia Mill. (USA): Smith College Botanical Garden, Northampton, USA – 2008 D. innoxia Mill. (SLO): Hortus Botanicus C. R. Labacensi, Slovenia – 2009 D. metel L. (PL): Department of Medicinal Plants, Poznan, Poland – 2007 D. metel L. (HU): Medicinal Plant Garden, University of Pećs, Hungary – 2008 D. meteloides (JAP) Medicinal Plant Garden, Tokyo, Japan – 2009 D. tatula (JAP) Medicinal Plant Garden, Tokyo, Japan – 2009 Plants were reared in the Botanical Garden (2007) or the Medicinal Plant Garden (2008, 2009) of the University of Pećs. Nectar samples were collected with

calibrated micro pipettes (DURAN) throughout the flowering period, during summer of 2007–2009. If the nectar content of a single flower did not reach 50 lL, samples from various flowers of the same species and same day were pooled. On July 17, 2009 the flowers of D. innoxia produced high amounts of nectar, which allowed the separate analysis of the alkaloid content in each flower. Samples were stored in Eppendorf tubes at -20 °C until further analysis.

Standard Solutions and Sample Preparation From methanolic stock solution of atropine and scopolamine (each 10 mg mL-1), standard solutions were prepared for the calibration. The standards were dissolved with eluent A (2% (v/v) formic acid in

150000 290

[M+H]+

Intensity

125000

100000

75000

50000

[M+Na]+ 25000

[M+K]+ 0

54

124 141 112

77 89

50

100

261 284

187 207 231

150

200

250

313

329 350

300

383 403 420

350

400

451 474 495 514528 550 567 590

450

500

550

m/z

Fig. 3. ESI-MS spectrum of atropine (solution at 10-2 mg mL-1 in methanol)

304

400e3

350e3

300e3

Intensity

[M+H]+ 250e3

200e3 156

150e3

138

100e3

[M+Na]+

121

50e3

0e3

59 7079

50

168 191205 221 243

115

100

150

200

250

296

326 338 358 377

402

350

400

300

430445

450

474 494

523

500

548

575 597

550

m/z

Fig. 4. ESI-MS spectrum of scopolamine (solution at 10-2 mg mL-1 in methanol)

methanol:water = 7:93). The solutions were stored at 4 °C and were used for a week. Before injections, the Datura samples were diluted (according to need) with eluent A, then filtered through a 0.45 mm pore size Syringeless filter (Mini-Uniprep, Whatman) and the injection volume was 2 lL.

Shimadzu), micro vacuum degasser (DGU-14 A, Shimadzu), system controller (SCL-10 AVP, Shimadzu), MS detector (LCMS-QP 8000 Shimadzu) and injector (7725i, Rheodyne) with 2 lL loop. LCMSsolution (Shimadzu) software was used to control the LC-MS system and for data processing. Chromatographic separations were performed on an Ascentis Express C18 column (50 9 2.1 mm, 2.7 lm, Supelco, USA) packed by 2.7 lm fused core particles that comprise a 1.7 lm solid core and a 0.5 lm porous shell. For the separations, a gradient of mobile phase A—2 v/v% formic-acid in methanol:water = 7:93—and mobile phase B—2 v/v% formic-acid in methanol—was used. The gradient profile was set as follows: 0.00 min 0% B eluent, 10.00 min 90% B eluent, 10.01 min 0% B eluent and 15.00 min 0% B eluent. The flow rate was 0.2 mL min-1, the column temperature was ambient. The column outlet was connected to the electrospray sample inlet. The electrospray source was operated in positive mode and the interface conditions were as follows: capillary voltage of 5 kV, CDL voltage of 5 V, CDL temperature of 250 °C and deflector voltage of 40 V. The desolvation gas flow was 3.5 L min-1, and was obtained from a nitrogen generator. The detector voltage was 2.0 kV. Mass spectrometer conditions (cone and collision energy) were optimized by direct infusion of the standards. SIM acquisition mode was used for the analysis, in order to detect only specific mass ions during the analysis.

LC-MS Instrumentation and Conditions The concentration of atropine and scopolamine was determined by LC-MS. The LC-MS system consisted of a liquid chromatograph (LC-10 ADVP,

Results and Discussion Determination of LOD, LOQ and Calibration Range The LOD was determined experimentally, and taken as the concentration producing a detector signal that could be clearly distinguished from the baseline noise (3 times baseline noise). The limit of quantitation (LOQ) was taken as the concentration that produced a detector signal ten times greater than the baseline

Original

a

290.00(1.00) 304.00(2.60)

500e3

Atropine tR = 5.4 min

400e3

Intensity

noise. A calibration curve was prepared at six different concentrations of atropine and scopolamine standard solutions in the concentration range of 1–100,000 ng mL-1. All injections were repeated three times (n = 3). The calibration ranges adequately covered the variations in the amounts of atropine and scopolamine in the samples. The correlation coefficients (r2) were 0.9995 and 0.9998, respectively. The LOD and LOQ values are summarized in Table 1.

300e3

Scopolamine tR = 4.1 min m/z = 290

200e3 100e3

m/z = 304 0e3 2.5

5.0

7.5

10.0

Time (min)

b

Repeatability and Intermediate Precision

LC-MS Analysis Results The MS spectrum of scopolamine and atropine is shown in Figs. 3 and 4. Each spectrum revealed a base peak at m/z 304 and 290, respectively, corresponding to the pseudo molecular ions [M+H]+. The fragment ions of scopolamine were observed at m/z 138 and 156, and at m/z 124 for atropine. The scopolamine has an epoxy group on the tropane ring and this difference causes the different fragmentation of both substances [31]. Datura floral nectar samples were measured (Fig. 5) directly without sample preparation. The results of LC-MS identification of the alkaloids content in Datura nectar samples are very different, depending on the species and date of sample collection (Table 6). Original

600e3

Intensity

500e3

Atropine tR = 5.4 min

Scopolamine tR = 4.1 min

400e3 300e3

m/z = 290

200e3 100e3

m/z = 304 0e3 2.5

5.0

7.5

10.0

Time (min)

c

290.00(9.71) 304.00(1.00)

750e3

Intensity

The retention time of atropine and scopolamine was observed to be 4.1 and 5.4 min, respectively. Total time of analysis was less than 15 min. Repeatabilities (intra- and inter-day precision) of the method were evaluated by assaying six replicate injections of a standard solution and a Datura nectar sample. The mean values and standard deviations of retention time and the concentration of atropine and scopolamine (from two Datura nectar samples) are listed in Tables 2, 3, 4, 5. The standard deviations proved the precision and the repeatability of the retention time to be very good.

290.00(1.25) 304.00(1.00)

500e3

Atropine tR = 5.4 min m/z = 290

250e3

Scopolamine tR = 4.1 min m/z = 304

0e3

2.5

5.0

7.5

10.0

Time (min)

Fig. 5. SIM chromatogram of scopolamine and atropine in D. tatula (a) D. innoxia (b) and D. metel (c) nectar samples

Scopolamine was the dominant alkaloid in all but one of the Datura nectar samples, while atropine was present only in trace amounts. The significantly higher ratio of scopolamine in the nectar was in accordance with data measured in all aerial parts (vegetative and reproductive organs alike) of D. me-

tel [15, 19], the roots and aerial parts of D. innoxia [17], as well as the flowers of D. arborea [13]. Despite the fact that in Datura flowers the nectar secreting tissues are localized around the ovary, an inverse alkaloid ratio was observed in the seeds of D. innoxia [1] and D. stramonium, the alkaloid content ranging

Table 6. Alkaloid content of different Datura floral nectar samples Sample

Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura Datura

Scopolamine

innoxia (CH) 2007 innoxia (PL/1) 2007 innoxia (PL/2) 2007 innoxia (USA) 2008 innoxia (SLO) 13 July 2009 innoxia (SLO) 15 July 2009 innoxia (SLO) 17 July 2009 - flower 1 innoxia (SLO) 17 July 2009 - flower 2 innoxia (SLO) 17 July 2009 - flower 3 innoxia (SLO) 17 July 2009 - flower 4 innoxia (SLO) 17 July 2009 - flower 5 innoxia (SLO) 17 July 2009 - flower 6 metel (PL) 2007 metel L. (HU) 2008 meteloides (JAP) 17 July 2009 meteloides (JAP) 11 September 2009 tatula (JAP) 06 August 2009

from 1.69 to 2.71 mg g-1 for atropine and 0.36–0.69 mg g-1 for scopolamine from different areas of the United States [12]. From the studied Datura species only D. tatula exhibited a nectar alkaloid profile where atropine was present in a higher amount than scopolamine. However, the measurements should be repeated in the next growing season, since in 2009 only a single, pooled nectar sample was available, due to the smaller size (75–120 mm corolla length) and therefore lower nectar quantities of the flowers. The other outstanding species was D. meteloides, where the scopolamine/ atropine ratio was significantly higher (452.05 and 631.89 on July 17 and September 11, respectively) than in other Datura species, on both sampling dates. Since the nectar samples of D. innoxia and D. metel showed rather similar alkaloid ratios, ranging from 6.45 to 30.98, the more pronounced dominance of scopolamine in the nectar appears to be a species characteristic for D. meteloides. Nectar samples from different flowers (individuals) of D. innoxia collected on the same day showed no significant differences concerning the ratio of the two alkaloids, however, the absolute amounts of scopolamine and atropine varied.

Atropine


RSD%


RSD%

226.46 62.00 400.04 199.80 157.20 57.80 328.20 294.40 227.00 148.40 342.50 133.90 84.57 131.66 189.86 120.06 11.00

1.32 2.47 3.39 2.63 1.97 5.60 0.98 2.72 5.11 1.75 4.73 4.18 1.74 1.57 1.53 1.00 2.73

1.79 3.38 3.30 2.15 5.70 3.43 30.00 31.70 22.30 23.00 37.00 10.60 3.25 4.25 0.42 0.19 17.60

5.59 4.73 4.55 6.51 4.04 2.92 4.67 3.79 1.79 3.91 5.95 4.72 2.77 4.47 4.76 5.26 3.24

Nectar Volumes and Alkaloid Levels in Relation to Toxicity Our data of 3 years (2007–2009) show that nectar volumes per flower vary considerably, ranging from 1.1 to 275.1, 1.5 to 143.7 and 51.3 to 298.1 lL/flower and averaging 112.61, 71.38 and 115.82 lL/flower in D. innoxia, D. metel and D. meteloides, respectively (unpublished data). The usual adult therapeutic doses are 0.5–1.0 and 0.4–0.8 mg of atropine and scopolamine, respectively, if administered orally. The toxic dose of atropine appears to vary considerably with individuals, but already 5–10 mg may produce symptoms like tachycardia, mydriasis, blurring of vision, ataxia, hallucination, delirium and coma. The lowest ingested dose of scopolamine associated with severe symptoms was 2–4 mg. Fatalities are reported to have occurred with doses of 50–100 mg of atropine, and for children the lethal dose of atropine and scopolamine alike may be as low as 10 mg [45]. Taking an average D. innoxia flower that produces 0.1 mL nectar with the average alkaloid (scopolamine) concentration of 215 lg mL-1, the consumption of the nectar of 100 flowers would suffice to induce severe symptoms of anticholinergic intoxication.

Conclusion A liquid chromatography method coupled with electrospray mass spectrometric detection for the determination of atropine and scopolamine in Datura floral nectar has been developed. The method needs no sample preparation for the separation prior to analysis. The results showed that the technique is very sensitive, reproducible, and accurate. The LOD for this method using ESI-MS detection is 75.08 pg mL-1 for atropine and 150.15 pg mL-1 for scopolamine. Our data confirmed that the alkaloid characteristics for the vegetative and reproductive parts of Datura plants are also present in the nectar secreted by the flowers, although in lower concentrations. In largeflowered (200–220 mm corolla length) Datura species (such as D. innoxia, D. metel and, D. meteloides), where the amount of nectar may reach 100–150 lL per flower, the alkaloid content increases proportionately and thus the nectar may be a potential source of intoxication.

Acknowledgments The work was supported by the grants GVOP-3.2.1-0168, RET 008/2005, OTKA-NKTH NI-68863, OTKA

Original

K75717, OTKA F48815. Simkon Ltd. (Budapest, Hungary) and Sigma-Aldrich Ltd. (Budapest, Hungary) are thanked for lending the Ascentis Express C18 column.

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