Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method

Accepted Manuscript Title: Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method Author: Szymon Dziomba Teresa Łepek Zbigniew Jaremicz Maria Łuczkiewicz Adam Prahl Piotr Kowalski PII: DOI: Reference:

S1570-0232(15)30067-2 http://dx.doi.org/doi:10.1016/j.jchromb.2015.06.029 CHROMB 19523

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

5-5-2015 24-6-2015 28-6-2015

Please cite this article as: Szymon Dziomba, Teresa Lepek, Zbigniew Jaremicz, Maria Luczkiewicz, Adam Prahl, Piotr Kowalski, Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.06.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method. Szymon Dziomba1, Teresa Łepek2, Zbigniew Jaremicz3, Maria Łuczkiewicz3, Adam Prahl2, Piotr Kowalski1 * 1

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Gdańsk, 107 Hallera Street,80-416 Gdańsk, Poland

2

Institute of Organic Synthesis, Department of Organic Chemistry, Faculty of Chemistry, University of Gdańsk, 63 WitaStwosza Street, 80-952 Gdańsk, Poland

3

Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Gdansk, 107 Hallera Street, 80-416, Gdańsk, Poland

Corresponding author: Tel.: +48 58 349 31 36, e-mail: [email protected] Highlights Simple method development process resulted in extremely rapid separationof selected tropane alkaloidswith very high efficiencywas developed. Quantification of scopolamine, hyoscyamine and anisodamine in plant cultures growth media without any pretretment step was presented. Impact of frequency of detector probing on the peaks parameters in CE has been demonstrated and discussed for the first time.

Abstract An electrophoretic method for fast separation of three tropane alkaloids (hyoscyamine, anisodamine and scopolamine) was presented. The substances were complete resolved in less than one minute due to utilization of relatively short capillary (20.2 cm effective length) and high voltage (25 kV). Detector probing frequency was found among the parameters that significantly affected the detection sensitivity. The performed experiments showed insufficient available probing frequency of used commercial spectrophotometric detector according to capillary electrophoresis (CE) separation potential. Under the optimized conditions the background electrolyte (BGE) was composed of 20 mMTris, 6 mMHCl and 20 mMNaCl (pH 8.50).All analyses were carried out in fused silica capillaries of 50 µm (inner diameter) and 31.2 cm (total capillary length). Samples were 1

injected hydrodynamically (5 s; 3.45 kPa) without any sample preparation step and separation was performed at 25 kV. The elaborated method was applied in plant cultures growth media analysis after incubation with hairy roots of selected Solanaceae species. The performed experiments proved the usefulness of CE in quality control of biotechnological processes.

Keywords:anisodamine; biotechnology; capillary zone electrophoresis; hyoscyamine; scopolamine; tropane alkaloids.

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1. Introduction Tropane alkaloids, such as hyoscyamine, scopolamine and anisodamine are important plant secondary metabolites which, because of their anti-cholinergic activity, are widely used in medicine as anti-spasmodics, mydriatics and to prevent motion sickness [1]. Due to the stereochemistry of tropane alkaloids, chemical synthesis of this compounds have proved economically unfeasible, therefore they are still obtained by extraction from plant material grow in field conditions what is rather problematic method of secondary metabolites production. Hence, for many years effort have been made to developed methods for obtaining tropane alkaloids in plant in vitro cultures. Experiments were conducted on different types of in vitro cultures of many Solanaceae plants, both in laboratory and bioreactor scale, but no commercially viable system have been yet obtained [2]. Biotechnological processes require constant monitoring of crucial physicochemical process parameters in real time to avoid making systematic errors or manual steps of the analysis procedures [3]. The most important measurements during the bioprocess are changes in pH and temperature and the concentrations of precursors and final products in biomasses and growth media [4]. It is essential to choose the appropriate conditions to optimize the regain process of products with a good amount and quality [5]. Breakdown of bioprocess monitoring techniques depends on the location of the sensor, which can be located inside (in situ technique) or outside of the instrument (ex-situtchnique), then the material is collected and analyzed on-line [6]. Hitherto the most popular analytical techniques used for the analysis of bio-products were gas chromatography (GC), liquid chromatography (LC) and spectroscopy [3; 7; 8], which are a time-consuming and relatively expensive in exploitation [7]. Capillary electrophoresis (CE) is an electrokinetic process wherein the electriccharged molecules move, as a result of the applied electric field, toward the electrode of opposite sign. Particular molecules move at different velocities, depending on their differential electrophoretic mobility as well as type, concentration and pH value of the background electrolyte (BGE) [9]. On the basis of these properties fast separation and characterization of various molecules in bioprocesses can be performed [10; 11]. In the previous experiments conducted on Hyoscyamusniger hairy root cultures in different bioreactor installations, compounds biosynthesized in biomass were partly released to the growth media. Concentrations of scopolamine, hyoscyamine and its precursor anisodamine in medium varied during the growth cycle. The highest amounts of abovementioned compounds were determined on the end of the experiments which corresponded to the decline growth 3

phase[12]. Therefore, tracking changes in products and precursor concentration in growth medium can be useful for monitoring of system productivity and the biomass growth cycle. A number of methods concerning separation of hyoscyamine, scopolamine and anisodamine have been published till now. Next to LC [13], GC [14] and thin layer chromatography [14], CE technique seems to be the most frequently used for simultaneous determination of these particular molecules [15; 16; 17]. Extensive discussion on the application of CE in alkaloids analysis can be found in the following reviews [14; 18; 19]. In brief, CE is becoming increasingly popular and widespread because of its advantages such as extremely high separation efficiency and resolution, low run cost and buffer consumption. Additionally, this technique features short analysis time and environmental friendliness, due to small solvents consumption [11; 20; 21; 22]. These advantages tend to the increased use of CE in the analysis of bioprocesses, for instance fermentation or cell cultures [8; 22]. The aim of the study was the elaboration of fast and reliable method for direct determination of hyoscyamine, anisodamine and scopolamine in plant cultures growth media. Simple method development process resulted in extremely rapid separation of the compounds of interest with very high separation efficiency. The CE method was successfully applied for simultaneous quantification of tropane alkaloids in a broth media of plant cultures.

2. Experimental 2.1. Apparatus All measurements were conducted using MDQ Capillary Electrophoresis System (Beckman Instruments, Fullerton, CA, USA) equipped with UV detector. Data were recorded at 200 nm with maximum available probing frequency (32 Hz). Separations were performed in uncoated fused silica capillaries (Beckman) of 50 µm x 31.2 cm (20.2 cm to detection window) termostated at 25 (±0.1 °C) unless otherwise stated. 2.2. Reagents and solutions 2-Amino-2-hydroxymethylpropane-1,3-diol (Tris; >99.8 %) was obtained from Bio-Rad Laboratories (Hercules, California, USA). Sodium chloride and hydrochloric acid were purchased from STANLAB (Lublin, Poland) and POCH (Gliwice, Poland). All standards were of analytical grade: hyoscyamine (L-atropine) (Extrasynthese, Genay, France), scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO, USA) and anisodamine (LKT Laboratories, St. Paul, MN, USA). All stock solutions (0.2 M Tris, 0.1 M HCl and 0.5 M NaCl) were prepared through dissolution of proper amount of substance in deionized water (Basic 5, Hydrolab, Wislina, 4

Poland). Standards were prepared in a concentration of 1 mg/ml in deionized water except hyoscyamine, which was prepared in 0.02 M HCl solution and they were in fridge before use. It should be stressed that anisodamine solution was freshly prepared every week while other solution were prepared every three weeks. Under the optimized conditions the BGE was composed of 20 mMTris, 6 mMHCl and 20 mMNaCl (pH 8.50). Desired pH of BGE was obtained with 0.2 M Tris or 0.1 M HCl solutions. The pKa values of the hyoscyamine (9.98 ± 0.40), scopolamine (8.01 ± 0.40) and anisodamine (8.94 ± 0.60) were calculated using ACD/ChemSketch software version 12.01 (Advanced Chemistry Development, Inc., Toronto, ON, Canada). 2.3. General electrophoresis procedure Conditioning of capillaries at the beginning and at the end of every working day was performed with 0.1 M NaOH, deionized water and BGE, each rinsed for 10 min at 70 psi (482.6 kPa) pressure. Before each separation, the capillary was conditioned for 2 min (50 psi ≈ 344.7 kPa) with BGE. Afterwards, capillary ends were dipped in water to prevent a carryover of buffer on the outer surface of the capillary. Next, the injection of sample was performed (5 s, 0.5 psi ≈ 3.45 kPa) followed by short plug of BGE (5 s, 0.5 psi). Then, high voltage was applied (25 kV, 0.5 min ramp time) and the separation was carried out. During analysis the current was in a range of 55-57 μA. Finally, the capillary was rinse with BGE (1 min, 70 psi). 2.4. Analytical material For

the

analysis

growth

media

collected

from

2-month

old

culture

of

HyoscyamusnigerandAtropabaetica were used. Hairy root cultures of H. nigerandA. baeticawere established as descried elsewhere [12; 23]. Both cultures were maintained in the dark, at 24 ± 1 °C, in 125 ml Erlenmeyer flasks (shaken at 120 rpm, 25.4 mm stroke) containing 25 ml phytohormone-free Murashige and Skoog (MS) medium (pH 6.0) supplemented with 3% (w/v) sucrose. The samples were analysed by using CE without any pretreatment step.

5

3. Results and discussion 3.1. Method optimization Highly similar chemical structure of determined compounds usually results in close values of their absolute electrophoretic mobilities. In such cases electrophoretic mobilities difference can be achieved by pH value adjustment of BGE near the pKa’s of analytes (calculated pKa values of alkaloids can be found in Section 2.1). According to this fact the baseline separation of alkaloids of interest was obtained using Tris buffer (pH 8.50). Next to desired pH value of BGE, the ionic strength of electrolyte had significant impact on the separation efficiency and resolution of peaks (Figure 2). Low ionic strength of 20 mMTris buffer solution resulted in incomplete separation of analytes (Figure 2A) while addition of NaCl to BGE noticeably improved both peaks efficiency and height (Figure 2B). Increase of the ionic strength with higher concentration of Tris instead of the addition of NaCl to 20 mMTris solution provided similar effect. However, Tris+ ions more efficiently suppress the electroosmotic flow (EOF) in capillary than Na+, thereby extending the time of analysis. The influence of the ionic strength of the BGE is especially important in discussed case where direct analysis of biological samples was intended. Relatively high amount of inorganic species in analyzed samples counteracted the stacking of analytes on the boundary of sample and BGE zones in capillary (Figure 2A) [24]. Increase of the BGE ionic strength successfully helped to overcome this inconvenience (Figure 2B). Fast separation of analytes in CE can be obtained by the application of short capillaries and high electric field strengths [25]. However, initially performed method development revealed significant relationship between the applied electric field during analysis and peaks intensity (Figure 3, squares and diamonds). In the experiment the highest sensitivity of the method was demonstrated for the smallest applied voltage (3 and 5 kV, which corresponds to 96 and 160 V/cm, respectively) and considerable decrease of these parameters with the voltage increase. The obtained results are in contradiction with the fundamental laws of CE, which states that less diffused peaks are expected if their migration time is shorter. The observed effect was due to the insufficient probing frequency (4 Hz) of the UV detector. Increase of the detector probing frequency from 4 to 32 Hz proved the falseness of the obtained relationship and highlights the importance of this factor in fast CE analysis (Figure 3). It is noteworthy that available maximal detector probing frequency (32 Hz) was found insufficient to maintain sensitivity as higher separation voltage (30 kV) and short end injection (effective capillary length: 11 cm) could have been applied providing faster analysis but also peaks of smaller intensity than the observed under the optimized conditions (25 kV, effective capillary length: 6

20.2 cm). Nevertheless, the complete separation of selected alkaloids in less than 1 minute was achieved due to utilization of the possibly shortest capillary for the device used in experiments (according to CE system vendor guidance it is 31.2 cm total capillary length) and application of 25 kV during analysis. The applied voltage generated current ranging from 55 to 57 µA which guaranteed the efficient cooling of the capillary (CV values of migration times for every 6 subsequent runs were below 0.4 %). 3.2. Validation study Whole analytical method was validated in terms of selectivity, linearity, limits of detection and quantification (LOD and LOQ, respectively) and accuracy by carrying out the recovery and precision studies. The selectivity of the method was assessed on the basis of analysis of blank growth media. During the study no interferences in the electrophoretic view were observed. According to the utilized capillary rinsing and water dipping procedures, no carryover effect was observed. The linearity of the method was assessed in the range from 5 to 100 µg/mL. Calibration curves were prepared using seven concentration levels (5, 20, 40, 55, 70, 85 and 100 µg/mL) by plotting the analyte concentration on the x-axis and the corrected peak area rations on the y-axis.. For each calibration point analysis of six independently prepared samples was performed. Applied linear regression model provided high determination coefficients values (R2 > 0.9987). Moreover, precision of each point and back-calculated concentrations of analytes meet the requirements of European Medicines Agency for bioanalytical method validation [26]. The LOD and LOQ values were calculated within the concentration corresponding to signal-to-noise ratio equal to 3 and 10, respectively (Tab. 1). The received in our work LOD and LOQ values are comparable with those obtained by other CE methods with employing a UV detector [16; 17]. Intra-day and inter-day accuracy of the method was evaluated for four different analytes concentrations (5, 15, 50 and 80 µg/mL). Six independently prepared samples were analyzed for each concentration level in intra-day assay while nine samples were analyzed during three consecutive days (three samples per day) in inter-day tests. Both intra- and inter-day experiments showed satisfactory precision and recovery for the elaborated CE method.

4. Application The optimized method was applied for determination of hyoscyamine, anisodamine and scopolamine in plant culture growth media samples after incubation of Solanaceae roots. A 7

detailed description of culturing conditions can be found in Section 2.4. The exemplaryelectropherograms of these analyses were presented in Figure 4. In all assayed samples analytes were completely resolved from matrix components enabling simultaneous determination of these three compounds in less than 1 min. Scopolamine was found in Atropabeatica samples while hyoscyamine and anisodamine were determined in Hyoscyamusniger. Moreover, scopolamine was detected in the sample from H.niger, but a level not exceeding the LOQ. In the remaining samples, tropane alkaloids were not identified. The detailed results of alkaloid analysis can be found in Table 2.

5. Conclusion The presented method enabled rapid separation and simultaneous determination of hyoscyamine, anisodamine and scopolamine in the plant culture growth media. Best to the authors knowledge it is the fastest method for separation of these three compounds published to date [13; 15; 16; 17; 27]. The frequency of detector probing was shown to have a significant impact on the obtained sensitivity parameters and in case of utilized instrument it limited the rapidity of the method. Moreover, the direct determination of the compounds of interest has shown the potential of electrophoretic techniques in this field. The advantage of this fact can be taken for microfluidic devices coupling with bioreactors for on-line monitoring of biosynthesis which application has not been presented yet [28].

Acknowledgements This work was supported by the Polish National Science Centre with funds granted based on the decision number DEC-2012/07/N/ST4/00356.

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References [1] G. Grynkiewicz, and M. Gadzikowska, Tropane alkaloids as medicinally useful natural products and their synthetic derivatives as new drugs, Pharmacol. Rep. 60 (2008) 439-63. [2] J. Palazon, A. Navarro-Ocana, L. Hernandez-Vazquez, and M.H. Mirjalili, Application of metabolic engineering to the production of scopolamine, Molecules, Switzerland, 2008, pp. 1722-42. [3] H. Turkia, S. Holmström, T. Paasikallio, H. Sirén, M. Penttilä, and J.-P. Pitkänen, Online Capillary Electrophoresis for Monitoring Carboxylic Acid Production by Yeast during Bioreactor Cultivations, Anal. Chem. 85 (2013) 9705-9712. [4] S. Ehala, I. Vassiljeva, R. Kuldvee, R. Vilu, and M. Kaljurand, On-line coupling of a miniaturized bioreactor with capillary electrophoresis, via a membrane interface, for monitoring the production of organic acids by microorganisms, Fresenius J. Anal. Chem. 371 (2001) 168-173. [5] H. Tahkoniemi, K. Helmja, A. Menert, and M. Kaljurand, Fermentation reactor coupled with capillary electrophoresis for on-line bioprocess monitoring, J. Pharma. Biomed. Anal. 41 (2006) 1585-1591. [6] C. Dietzsch, O. Spadiut, and C. Herwig, On-line multiple component analysis for efficient quantitative bioprocess development, J. Biotechnol. 163 (2013) 362-370. [7] H. Turkia, H. Sirén, M. Penttilä, and J.-P. Pitkänen, Capillary electrophoresis for the monitoring of phenolic compounds in bioprocesses, J. Chromatogr. A 1278 (2013) 175-180. [8] J.M. Schrickx, M.J.H. Raedts, A.H. Stouthamer, and H.W. Vanverseveld, Organic Acid Production by Aspergillus niger in Recycling Culture Analyzed by Capillary Electrophoresis, Anal. Biochem. 231 (1995) 175-181. [9] M. Chiari, N. Dell'Orto, and L. Casella, Separation of organic acids by capillary zone electrophoresis in buffers containing divalent metal cations, J. Chromatogr. A 745 (1996) 93101. [10] M.E. Roche, R.P. Oda, and J.P. Landers, Capillary Electrophoresis in Biotechnology, Biotechnol. Prog. 13 (1997) 659-668. [11] H. Turkia, H. Sirén, J.-P. Pitkänen, M. Wiebe, and M. Penttilä, Capillary electrophoresis for the monitoring of carboxylic acid production by Gluconobacter oxydans, J. Chromatogr. A 1217 (2010) 1537-1542. [12] Z. Jaremicz, M. Luczkiewicz, A. Kokotkiewicz, A. Krolicka, and P. Sowinski, Production of tropane alkaloids in Hyoscyamus niger (black henbane) hairy roots grown in bubble-column and spray bioreactors, Biotechnol. Lett. 36 (2014) 843-53. 9

[13] P. Zhang, Y. Li, G. Liu, X. Sun, Y. Zhou, X. Deng, Q. Liao, and Z. Xie, Simultaneous determination of atropine, scopolamine, and anisodamine from Hyoscyamus niger L. in rat plasma by high-performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetics study, J. Sep. Sci. 37 (2014) 2664-74. [14] B. Dräger, Analysis of tropane and related alkaloids. J. Chromatogr. A 978 (2002) 1-35. [15] B. Yuan, C. Zheng, H. Teng, and T. You, Simultaneous determination of atropine, anisodamine, and scopolamine in plant extract by nonaqueous capillary electrophoresis coupled with electrochemiluminescence and electrochemistry dual detection, J. Chromatogr. A 1217 (2010) 171-174. [16] N. Ye, R. Zhu, X. Gu, and H. Zou, Determination of scopolamine, atropine and anisodamine in Flos daturae by capillary electrophoresis, Biomed. Chromatogr. 15 (2001) 509-512. [17] N. Ye, J. Li, C. Gao, and Y. Xie, Simultaneous determination of atropine, scopolamine, and anisodamine in Flos daturae by capillary electrophoresis using a capillary coated by graphene oxide, J. Sep. Sci. 36 (2013) 2698-702. [18] E. Aehle, and B. Dräger, Tropane alkaloid analysis by chromatographic and electrophoretic techniques: An update, J. Chromatogr. B 878 (2010) 1391-1406. [19] S. Dziomba, M. Belka, P. Kowalski, A. Plenis, and T. Baczek, The advances of electromigration techniques applied for alkaloid analysis, Biomed. Chromatogr. 27 (2013) 1312-1338. [20] J. He, S. Chen, and Z. Yu, Determination of poly-β-hydroxybutyric acid in Bacillus thuringiensis by capillary zone electrophoresis with indirect ultraviolet absorbance detection, J. Chromatogr. A 973 (2002) 197-202. [21] J. He, X. Luo, S. Chen, L. Cao, M. Sun, and Z. Yu, Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis, J. Chromatogr. A 994 (2003) 207-212. [22] J. Kittell, B. Borup, R. Voladari, and K. Zahn, Parallel capillary electrophoresis for the quantitative screening of fermentation broths containing natural products, Metab. Eng. 7 (2005) 53-58. [23] R. Zarate, N. el Jaber-Vazdekis, B. Medina, and A.G. Ravelo, Tailoring tropane alkaloid accumulation in transgenic hairy roots of Atropa baetica by over-expressing the gene encoding hyoscyamine 6beta-hydroxylase, Biotechnol. Lett. 28 (2006) 1271-7. [24] F.E.P. Mikkers, F.M. Everaerts, and T. Verheggen, Concentration distributions in free zone electrophoresis, J. Chromatogr. 169 (1979) 1-10. 10

[25] F.M. Matysik, Advances in fast electrophoretic separations based on short capillaries, Anal. Bioanal. Chem. 397 (2010) 961-965. [26] http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC5 00109686.pdf. [27] Z. Jaremicz, M. Luczkiewicz, M. Kisiel, R. Zárate, N.E. Jaber-Vazdekis, and P. Migas, Multi-development–HPTLC Method for Quantitation of Hyoscyamine, Scopolamine and their Biosynthetic Precursors in Selected Solanaceae Plants Grown in Natural Conditions and as In Vitro Cultures, Phytochem. Anal. 25 (2014) 29-35. [28] E.R. Castro, and A. Manz, Present state of microchip electrophoresis: State of the art and routine applications, J. Chromatogr. A 1382 (2015) 66-85.

Fig.1. Chemical structure of the analyzed alkaloids.

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Fig.2. Influence of ionic strength on the resolution of peaks. Conditions: BGE, (A) 20 mMTris, 6 mMHCl (pH 8.50); (B) the same as in (A) + 20 mMNaCl; applied voltage, 25 kV; capillary, 50 µm x 31.2 cm; sample injection, 5 s (0.5 psi); UV detection, 200 nm (32 Hz).

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Fig.3. The influence of applied electric field strength on obtained peaks intensity of hyoscyamine (black) and scopolamine (grey). The experiment was carried out using two different detector probing frequencies: squares and diamonds - 4 Hz; crosses and dashes- 32 Hz. Other conditions the same as in Figure 2.

13

14

Fig. 4.Exemplary electropherograms obtained from analysis of culturing media of hairy roots of (A) Atropa baetica and (B) Hyoscyamus niger. Other conditions the same as in Figure 2.

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Tab.1.Validation parameters determined for elaborated method. Abbreviations: CV – coefficient of variance. Hyoscyamine Linearityrange

Anisodamine

Scopolamine

5 – 100 µg/mL

Slope (a) Intercept (b) 2

Determinationcoefficient (R )

110.58

109.58

89.885

-90.158

-59.517

-18.677

0.9988

0.9987

0.9990

Limit of detection (LOD)

1.5 µg/mL

Limit of quantification (LOQ)

5.0 µg/mL

Separation efficiency(plates/m)*

383.8

451.6

761.3

Intra-day precision (CV; n=6) 5 µg/mL

6.3

8.8

5.2

15 µg/mL

8.3

7.0

7.4

50 µg/mL

5.7

3.2

5.7

80 µg/mL

6.8

3.1

7.0

Intra-day recovery (n=6) 5 µg/mL

103.1

92.5

113.1

15 µg/mL

107.2

93.1

104.0

50 µg/mL

95.4

90.1

99.9

80 µg/mL

99.8

88.2

102.9

Inter-day precision (CV; n=9) 5 µg/mL

5.9

8.6

7.5

15 µg/mL

8.3

5.6

7.6

50 µg/mL

4.9

3.3

4.9

80 µg/mL

5.5

3.7

5.4

Inter-day recovery (n=9) 5 µg/mL

104.2

90.6

111.7

15 µg/mL

102.7

92.3

104.5

50 µg/mL

99.4

89.6

102.4

80 µg/mL

97.9

89.5

101.0

* Separation efficiency was determined for six measurements of samples at concentration of each compound at 55 µg/mL.

16

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Tab.2. The results of the analysis of two different cultures growth media. Determined concentrations were expressed as the average concentration of substance ± expanded uncertainty [µg/mL] (for 95% level of confidence). Analytical media

Hyoscyamine

Anisodamine

Scopolamine

< LOD

< LOD

15.54 ± 0.59

80.46 ± 5.15

5.58 ± 0.39

< LOQ

Growth medium from A. baeticahairy roots cultures Growth medium from H.nigerhairy roots cultures

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