New Liquid Chromatography: Mass Spectrometry Assay for Natural Phytoestrogens from Vegetable Extracts

Acta Chromatographica 23(2011)3, 509–520 DOI: 10.1556/AChrom.23.2011.3.11 New Liquid Chromatography: Mass Spectrometry Assay for Natural Phytoestrogens from Vegetable Extracts

L. VLASE1, D.-S. POPA2,*, A. TERO-VESCAN3, AND N. OLAH4

1Department of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy, Emil Isac 13, RO-400023 Cluj-Napoca, Romania 2Department of Toxicology, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy, Emil Isac 13, RO-400023 Cluj-Napoca, Romania 3Department of Pharmaceutical Biochemistry, University of Medicine and Pharmacy, Gheorghe Marinescu 38, RO-540000 Targu-Mures, Romania 4Department of Drug Industry and Pharmaceutical Biotechnology, Faculty of Pharmacy, Vest University “Vasile Goldis” Arad and SC PlantExtrakt SRL, RO-407059 Radaia, Cluj, Romania E-mail: [email protected]

Summary. A new liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of seven isoflavones (daidzin, genistin, ononin, daidzein, glycitein, genistein, and formononetin) and coumestrol in vegetable extracts was developed. The separation was performed on a Zorbax SB-C18 column with a mixture of methanol (solvent A) and 0.1% (v/v) acetic acid in water (solvent B) under gradient conditions at 50°C with a flow rate of 1 mL min−1. The detection of analytes was performed by electrospray ionization, negative ionisation, in non-reactive MS2 mode for aglycons or in reactive MS2 mode for glycosides. The method shows a good linearity (r2 > 0.9948) over the concentration range of 40–4000 ng mL−1 for all analytes, a good precision (CV < 11%) and accuracy (<10%). The method was successfully applied to quantify the isoflavones and coumestrol in vegetable extracts obtained from red clover (Trifolium pratense L., Fabaceae) and dyer’s greenweed (Genista tinctoria L., Fabaceae) and can be used in the chemical characterization of vegetables with phytoestrogen content. Key Words: phytoestrogens, isoflavones, LC-MS/MS, Trifolium pratense, Genista tinctoria

Introduction

In recent years, the knowledge of the positive health effects of vegetables has been increasing. Isoflavones, lignans and coumestans are compounds with polyphenolic structures that exhibit estrogenic and anti-estrogenic ac- tivity, anti-oxidant and anti-carcinogenic properties, and protective effects against a number of complex diseases, such as cardiovascular disease, os- teoporosis and menopausal symptoms [1, 2]. Soybeans are the most impor- tant natural source of genistein (4′,5,7-trihidroxyisoflavone) and daidzein (4′,7-dihidroxyisoflavone), which occur mainly as the glycosides genistin and daidzin. Other sources such as red clover (Trifolium pratense L., Fa- baceae) are rich in other aglycones such as formononetin, biochanin A, or

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glycitein [1]. Red clover extracts offer the clinical benefits and represent an alternative to conventional hormone replacement therapy in menopausal disorders and hormone-dependent diseases [3]. Genista species (Fabaceae) show interesting biological properties such as hypoglycemic, anti- inflammatory, anti-ulcer, spasmolytic, anti-oxidant and estrogenic effects [4]. Dyer’s greenweed (Genista tinctoria L.) is rich in genistein [5, 6] and showed a protective effect against UV light, anti-oxidant activities and in- hibited the growth of melanoma cells in vitro [4]. There are many studies to quantify the phytoestrogen content in vegetable extracts or dietary supplements by HPLC [5, 7–10], GC-MS [11], and capillary electrophoresis [12, 13]. HPLC with UV detection is often chosen for routine analysis, but a preliminary acid or basic hydrolysis of isoflavone derivatives is required. The liquid chromatography tandem mass spectrometry (LC-MS/MS) assay offers considerable advantages by its powerful performances: speed, selectivity, sensitivity, and robustness. It is the method preferred to identify isoflavone derivatives based on the fragmentation pattern of the parent ion and in the quantification of isoflavones in complex mixtures [14]. The GC-MS methods required a supplementary step for derivatisation such as trimethylsilyl derivatives [11] that makes the analysis longer and more expensive. The aim of this work is to develop a new, simple, and efficient LC/MS- MS assay for the quantification of seven isoflavones (three glycosides: daidzin, genistin, and ononin, and four aglycones: daidzein, glycitein, genistein, and formononetin, respectively) and coumestrol (Fig. 1) from vegetable extracts of red clover (T. pratense L.) and dyer’s greenweed (G. tinctoria L.) from Transylvania area, Romania.

R1 R2 R3 R4 R1O O Daidzein H H H H Daidzin O-beta-D-GLU* H H H Genistein H H OH H R Genistin O-beta-D-GLU H OH H 2 Formononetin H H H CH3 Ononin O-beta-D-GLU H H CH3 R3 O Glycitein H OCH3 HH OR4 *GLU=glucosyl

HO O O

Coumestrol

O OH Fig. 1. Chemical structures of analyzed phytoestrogens

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Experimental

Reagents

Genistein (4′,5,7-trihydroxyisoflavone), genistin, daidzein, ononin, and coumestrol of HPLC grade were reference standards from Fluka (Steinheim, Germany). Glycitein, daidzin, and formononetin standards of analytical reagent grade were obtained from ChromaDex (the United States). Methanol of HPLC grade, acetic acid of analytical reagent grade, and 70% v/v ethanol of pharmaceutical grade were purchased from Merck KGaA (Darmstadt, Germany). Bidistilled, deionised water pro injections were purchased from Infusion Solution Laboratory of the University of Medicine and Pharmacy Cluj-Napoca (Romania).

Apparatus

The following apparatus were used: 204 Sigma Centrifuge (Osterode am Harz, Germany); Analytical Plus and Precision Standard Balance (Mettler- Toledo, Switzerland); Vortex Genie 2 mixer (Scientific Industries, New York); Ultrasonic bath Elma Transsonic 700/H (Singen, Germany). The HPLC system used was an 1100 series Agilent Technologies model (Darm- stadt, Germany) consisting of a G1312A binary pump, an in-line G1379A degasser, a G1329A autosampler, a G1316A column thermostat, and an Agilent Ion Trap Detector 1100 SL.

Chromatographic and Mass Spectrometry Conditions

Chromatographic separation was performed on a Zorbax SB-C18 (100 mm × 3.0 mm i.d., 5 μm) column (Agilent Technologies) equipped with a Zorbax SB-C18 precolumn with a mixture of methanol (solvent A) and 0.1% (v/v) acetic acid in water (solvent B) under gradient conditions (linear profile): 0 min—20% A, 2 min—20% methanol, 10 min—40% A, 10.5 min— 40% A, 11.5 min—45% A, 14.8 min—45% A, 15.8 min—100% A, at 50°C with a flow rate of 1 mL min−1. The detection of analytes was performed in non-reactive MS2 mode for the quantification of aglycons (only pseudo- molecular ion accumulation into ion trap, and then detection) or in reactive MS2 mode for the quantification of glycosides (accumulation and fragmentation of the pseudo-molecular ion, and then detection of fragments), negative ion fragmentation, using an ion trap mass spectrometer equipped with an electrospray ionisation (ESI) ion source: capillary +2500 V, nebulizer

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65 psi (nitrogen), dry gas nitrogen at 12 L min−1, and dry gas temperature 360°C.

Standard Solutions

The stock solutions of daidzin, genistin, ononin, daidzein, genistein, formononetin, glycitein, and coumestrol (1 mg mL−1) were prepared by dis- solving the appropriate quantities in methanol. Two working solutions (4000 μg mL−1 and 400 ng mL−1) were prepared by appropriate dilution in water. These solutions were used to prepare calibration standards with the concentrations of 40, 80, 160, 320, 480, 960, 1920, and 4000 ng mL−1. To ver- ify the precision and accuracy of the method, five control standards of 40 ng mL−1 (lower limit of quantification [LLOQ]), 320 ng mL−1 (medium level) and 1920 ng mL−1 (higher level) were prepared. The resultant calibration and control standards were preserved into 15 mL polypropylene tubes and stored at −4°C until analysis.

Vegetable Samples

The elaborated LC/MS-MS method was applied to quantify the eight poly- phenols in a tincture obtained from red clover (T. pratense L.) harvested from Cluj-Napoca area, Romania, and an extract obtained from dyer’s greenweed (G. tinctoria L.) harvested from Targu-Mures area, Romania. The T. pratense tincture was prepared from fresh herba using 14 g of 70% (v/v) ethanol to 10 g of fresh plant material, according to the 6th edition of the European Pharmacopoeia—mother tinctures for homeopathic preparations [15]. The active compounds were extracted by cold extraction (mac- eration). The vegetal product and solvent were let to stand for 10 days in a dark place, with a short gentle stirring once daily. The tincture was obtained by pressing the plant–ethanol mixture and then filtering it. The G. tinctoria extract was prepared from dry herba using 100 mL of 75% (v/v) methanol to 5 g of dry plant material. The active compounds were extracted by refluxing for 4 h at 60°C. The extract was obtained by filtration of the plant–methanol mixture and then by completion at 100 mL.

Quantification Method

The concentrations of the eight analytes were determined automatically by the instrument data system using peak areas and the external standard method. The calibration curve model was determined by the quadratic analysis: y = ax2 + bx + c, where y is the peak area and x is analyte concentration.

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The LLOQ was established as the lowest calibration standard with an accuracy and a precision <20%. The precision (expressed as coefficient of variation, CV, %) and accuracy (expressed as relative difference between obtained and theoretical concentration, bias, %) were determined for 40 ng mL−1 (LLOQ), 320 ng mL−1 (medium level) and 1920 ng mL−1 (higher level) by the analysis of five different standard solutions (n = 5) of these concentrations.

Results and Discussion

The isoflavones are the polar compounds that can be readily ionized in solution. For their analyses we used ESI. To obtain a good chromatographic separation of isoflavones by LC-ESI-MS-MS, an acidic eluent phase was employed. Bacaloni et al. [16] used a mobile phase with TFA and positive ionization (PI) for higher ionic signal intensities. Cuyckens and Claeys [17] reported that in PI mode, methanol containing 1% acetic acid was the most sensitive, whereas in the negative ionization (NI) mode, the highest sensitivity was obtained with a mobile phase containing 0.1% formic acid. The isoflavones have polyphenolic structures, and they can easily lose a proton to form the negative ions [M–H]−; however, they can also be detected in PI mode. Both NI and PI modes were tested for daidzin and daidzein as representative compounds (120 ng mL−1 each), and the signal-to-noise (S/N) ratio was calculated. The S/N ratio in NI was 8 and 2.5 times greater than that in PI for daidzin and daidzein, respectively. For this reason we preferred to use the NI. In our experimental conditions, methanol and 0.1% acetic acid give better chromatographic resolution and S/N ratios. To obtain a good analytical run time, the chromatographic separation was performed with mobile phase gradient. The retention times of all analyzed isoflavones are shown in Table I. Very little response was obtained using an atmospheric pressure chemical ionisation (APCI) interface, according to Bacaloni et al. [16]. APCI can be used for relatively non-polar compounds that can undergo acid–base reactions in the gas phase [18], and it is not preferred in the isoflavone analysis. For glycosides daidzin, genistin, and ononin, the fragmentation corre- sponds to the loss of a glucose molecule [M-162]− (m/z 253, 269 and 267). The conjugated compounds are by nature very fragile, but the remaining fragment is more resistant to further fragmentation. No fragmentation of aglycons molecules was obtained in either negative or positive mode. Their detection and quantification were performed in the non-reactive MS2 mode, whereas the aglycones daidzein, genistein, formononetin, and glycitein

Unauthenticated | Downloaded 10/02/21 08:52 AM UTC 514 L. Vlase et al. were detected and quantified in the reactive MS2 mode (m/z 253, 269, 267, and, 283) (Table I). Coumestrol and formononetin are isomer compounds; they have the same molecular mass and the same MS spectra of pseudo- molecular ion (Fig. 2). We used the same ion monitoring for their detection (m/z 267).

Intens. -MS2(415.0), 3.7-3.8min #(913-932) x106 252.8 0.5 415.0 0.0 x106 -MS2(431.0), 5.4-5.5min #(1279-1296) 1.0 431.0 268.9 0.5 310.9 0.0 x105 -MS2(429.0), 8.8min #2100 266.9

0 x106 -MS2(253.0), 9.1min #2179 2 252.9

0 x106 -MS2(283.0), 10.2min #2463 282.9 2

0 x106 -MS2(269.0), 11.0min #2712 268.9 4 2 0 x106 -MS2(267.0), 12.7min #3151 4 266.8

0 x106 -MS2(267.0), 14.4min #3516 266.9 4 2 0 240 260 280 300 320 340 360 380 400 420 m/z

Fig. 2. MS spectra of analyzed phytoestrogens (up to down, in the order of retention time: daidzin, genistin, ononin, daidzein, glycitein, genistein, coumestrol, and formononetin)

The selectivity of the method depends on chromatographic separation and mass spectrometry detection. A representative chromatogram of a standard solution with the eight analytes (320 ng mL−1) is shown in Fig. 3, and a chromatogram of red clover tincture analyzed Fig. 4.

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Intens. PROBACC___00007.D: TIC -All MSn x106 2

0 x106 PROBACC___00007.D: EIC 252.4-253.4 -MS2(415.0) 1.0 0.5

0.0 x106 PROBACC___00007.D: EIC 268; 269 -MS2(431.0)

0.5

0.0 x105 PROBACC___00007.D: EIC 266.4-267.4 -MS2(429.0)

0 x106 PROBACC___00007.D: EIC 252.4-253.4 -MS2(253.0)

0 x106 PROBACC___00007.D: EIC 282.4-283.4 -MS2(283.0) 2

1 0 x106 PROBACC___00007.D: EIC 268.4-269.4 -MS2(269.0)

0 x106 PROBACC___00007.D: EIC 266.3-267.3 -MS2(267.0)

0 2 4 6 8 10 12 14 Time [min]

Fig. 3. The chromatogram of a sample spiked with the eight analytes at medium-level concentration (320 ng mL−1), MS detection [up—TIC chromatogram with all compounds; in the order up to down the chromatograms of (2) daidzin (3.7 min); (3) genistin (5.5 min); (4) ononin (8.9 min); (5) daidzein (9.2 min); (6) glycitein (10.2 min); (7) genistein (11.0 min); (8) coumestrol (12.7 min); and (9) formononetin (14.4 min)]

The calibration curves were linear over the concentration range of 40– 4000 ng mL−1 for all analyzed compounds, with the correlation coefficients >0.9948 (Table II). The LLOQ was 40 ng mL−1 for all analytes. The values obtained for the precision and accuracy of curve calibrations at 40 ng mL−1 (LLOQ), 320 ng mL−1 (medium level), and 1920 ng mL−1 (higher level) are shown in Table II. All values for accuracy and precision were within the accepted limits (<10% and <11%, respectively).

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Table I. The retention times and the detection parameters (detection mode, m/z values) of the analyzed phytoestrogens

Retention Detection m/z parent m/z moni- No. Compound time (min) mode ion [M−H]− torised 1 Daidzin 3.7 R-MS2 415 253 2 Genistin 5.5 R-MS2 431 268, 269 3 Ononin 8.9 R-MS2 429 267 4 Daidzein 9.2 NR-MS2 253 253 5 Glycitein 10.2 NR-MS2 283 283 6 Genistein 11.0 NR-MS2 269 269 7 Coumestrol 12.7 NR-MS2 267 267 8 Formononetin 14.4 NR-MS2 267 267

R-MS2 = reactive MS2; NR-MS2 = non-reactive MS2

Table II. The precision (CV, %) and accuracy (bias, %) for the measurement of the eight analyzed phytoestrogens (n = 5) and the calibration parameters for the concentration range of 40–4000 ng mL−1 (N = 8, n = 5)

Nominal Found concentra- CV Bias Determination Compound concentration tion (%) (%) coefficient (r2) (ng mL−1) ng mL−1 ±SD 40.0 36.82 1.87 5.07 −7.94 Daidzin 320.0 344.77 8.93 2.59 7.74 >0.9997 1920.0 1773.87 65.64 3.70 −7.61 36.0 33.96 1.18 3.47 −5.66 Genistin 288.0 307.70 8.10 2.63 6.84 >0.9997 1728.0 1686.58 42.73 2.53 −2.40 38.4 35.33 1.94 5.50 −8.00 Ononin 307.2 337.07 23.31 6.92 9.72 >0.9999 1843.2 1840.16 43.44 2.36 −0.17 42.4 38.53 1.47 3.82 −9.13 Daidzein 339.2 367.89 10.48 8.46 2.85 >0.9984 2035.2 2011.21 134.56 6.69 −1.18 43.0 41.55 4.35 10.47 −3.38 Glycitein 344.0 359.82 19.41 5.39 4.60 >0.9974 2064.0 1938.59 207.80 10.72 −6.08 40.8 37.31 1.32 3.54 −8.56 Genistein 326.4 354.24 12.94 3.65 8.53 >0.9991 1958.4 1829.75 37.15 2.03 −6.57 41.6 39.75 4.00 10.05 −4.46 Coumestrol 332.8 331.79 15.45 4.66 −0.30 >0.9948 1996.8 1881.21 206.18 10.96 −5.79 41.2 39.15 4.14 10.56 −4.98 Formononetin 329.6 348.24 30.07 8.63 5.66 >0.9985 1977.6 1800.33 46.52 2.58 −8.96

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Intens. PROBA___000009.D: TIC -All MSn x106 4 2 0 x104 PROBA___000009.D: EIC 252.4-253.4 -MS2(415.0)

0 x105 PROBA___000009.D: EIC 268; 269 -MS2(431.0) 1

0 x104 PROBA___000009.D: EIC 266.4-267.4 -MS2(429.0)

0 x106 PROBA___000009.D: EIC 252.4-253.4 -MS2(253.0) 1.0

0.5

0.0 x106 PROBA___000009.D: EIC 282.4-283.4 -MS2(283.0) 1.0

0.5

0.0 x106 PROBA___000009.D: EIC 268.4-269.4 -MS2(269.0)

0 x106 PROBA___000009.D: EIC 266.3-267.3 -MS2(267.0) 6 4 2 0 0 2 4 6 8 10 12 14 Time [min]

Fig. 4. The chromatogram of the red clover tincture, MS detection [up—TIC chromatogram with all compounds; in the order up to down the chromatograms of (2) daidzin (3.7 min); (3) genistin (5.5 min); (4) ononin (8.9 min); (5) daidzein (9.2 min); (6) glycitein (10.2 min); (7) genistein (11.0 min); (8) coumestrol (12.7 min); and (9) formononetin (14.4 min)]

Our developed LC-MS/MS assay is simple, rapid, and accurate. In comparison with other published LC-MS [19–21] or HPLC-UV [14, 22, 23] assays for the quantification of isoflavones in vegetable extracts or dietary supplements, our method performs better in terms of speed (run time of only 15 min) and sensitivity (limit of quantification [LOQ] of 40 ng mL−1 for all analytes) (Table III).

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Table III. Some HPLC and LC-MS methods selected from literature applied to isoflavone separation

Run Analysed Analytical References Samples time LOQ analytes method (min) Different vege- Eight isofla- Antonelli LC-TISP- tables from vones and 28 2 μg g−1 et al. (2005) MS/MS, PI Leguminosae coumestrol Chen et al. Nutritional Nine isofla- LC-APCI- 48 500 ng mL−1 (2005) supplements vones MS, NI Delmonte Soy, red clover Twenty-one 90 NA HPLC-PDA et al. (2006) and kudzu isoflavones Schwartz Seven isofla- HPLC-UV- Foodstuffs 20 25 ng mL−1 et al. (2009) vones ESI-MS, PI Otieno et al. Eleven LC-ESI- Soymilk 23 NA [21] isoflavones MS/MS, PI Luthria et al. Twelve Soybean 44 NA HPLC-UV [22] isoflavones Visnevski Twelve 20– T. pratense 40 HPLC-PDA et al. (2009) isoflavones 200 ng mL−1 TISP = turbo ion spray; NA = not available

Depending on the origin, geographical and climate area, the various plant samples show variable contents of isoflavones. The values of isoflavone content determined in analyzed vegetable material (Table IV) harvested from Transylvania area (Romania) are in accordance with other data from scientific literature [3, 5, 14]. T. pratense has a high content in formononetin (25.47 mg per 100 g fresh vegetable material) and G. tinctoria in genistein (71.50 mg per 100 g dry vegetable material) and genistin (49.47 mg per 100 g dry vegetable material). Coumestrol and glycitein were not detected in the analyzed plants.

Table IV. The phytoestrogen content of vegetable extracts analyzed

Vegetable DZ GN ON DZE GLY GNE COU FOR extract

TPa 0.12 0.21 0.90 1.47 0.00 3.91 0.00 25.47 GTb 2.13 49.47 0.00 10.85 0.00 71.50 0.00 7.04

DZ = daidzin; GN = genistin; ON = ononin; DZE = daidzein; GLY = glycitein; GNE = genistein; COU = coumestrol; FOR = formononetin aT. pratense tincture (mg per 100 g fresh vegetable material) bG. tinctoria extract (mg per 100 g dry vegetable material)

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Conclusion

The developed LC-MS/MS method proves a good linearity (r2 > 0.9948) over the concentration range of 40–4000 ng mL−1 for all analysed isoflavones and coumestrol, a good sensitivity (LOQ of 40 ng mL−1 for all analytes), precision (CV < 11%), and accuracy (<10%). It is more rapid than other similar methods used for isoflavone analysis (run time of 15 min). It was successfully applied to quantify the isoflavones and coumestrol in vegetable extracts obtained from red clover (T. pratense L., Fabaceae) and dyer’s greenweed (G. tinctoria L., Fabaceae) harvested from Transylvania area (Romania) and can be used in the chemical characterization of vegetables or dietary supplements having phytoestrogen content.

Acknowledgments

This work was supported by the project PN-II-ID-PCE 1337/2008 financed by CNCSIS Romania.

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