ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2007, том 62, № 7, с. 745-749
^=ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
FAST QUANTIFICATION OF FLAVONOIDS IN FILIPENDULAE ULMARIAE FLOS BY HPLC/ESI-MS USING A NON POROUS STATIONARY PHASE
© 2007 r. E. Pemp, G. Reznicek, L. Krenn
Department of Pharmacognosy, University of Vienna, ParmaCenter Vienna Althanstr. 14, A-1090 Vienna, Austria Received 12.10.2005; in final form 17.05.2006
A sensitive and rapid method for the quantification of flavonoids in Filipendulae ulmariae flos (Filipéndula ulmaria L.) was developed using HPLC with diode array detection coupled to electrospray ionisation mass spectrometry (ESI-MS). Separation was achieved on a 1.5 |jm non-porous C-18 phase by gradient elution with acetonitrile and 0.03M acetic acid. The quantification was performed by internal standardisation with acacetin. To enhance selectivity, ESI-MS detection was optimized in negative mode and selected ion monitoring (SIM) mode.
Meadowsweet (Filipéndula ulmaria L.) is a common herbal plant in Europe. The dried flowers are traditionally used in supportive treatment of colds because of the diuretic and diaphoretic effects. Application in treatment of rheumatism and gout is also reported [1]. Pharmacological investigations showed antimicrobial and anti-inflammatory activities of ethanolic and aqueous extracts from meadowsweet flowers [2, 3]. A hep-arin-like complex found in Filipendulae ulmariae flos had anticoagulant and fibrinolytic effects in several in vivo experiments [4, 5]. Various extracts of meadowsweet flowers showed strong immunomodulatory activity towards the classical pathway of complement activation [6]. The flavonoid complex is considered to be responsible for the antiulcerogenic activity of the drug [7, 8].
Although there are many studies about the pharmacological effects of Filipéndula ulmaria, only few methods are available for the quality control of the drug. Quantitative analysis of meadowsweet flowers and herb in the French and European Pharmacopoeia is performed by the quantification of its essential oil, of which only low amounts (less than 0.5%) are detected in the drug [9, 10]. Pharmacological investigations show a significant contribution of flavonoids to the pharmacological effects of meadowsweet. Furthermore, in stability control of drugs and herbal medicinal products, analysis of different active components is requested. Thus, the quantification of flavonoids should be included in the quality and stability control of Filip-iendulae ulmariae flos.
The drug contains high amounts of flavonoids, up to 6%. Seven main flavonoids, spiraeoside, quercetin-3'-glucosid, avicularoside, hyperoside, rutin, kaempferol-4'-glucoside, and the aglycon quercetin, have been re-
ported for meadowsweet flowers (Fig. 1) [11, 12]. Quantification of these compounds is usually performed by HPLC on porous stationary phases and UV detection [13]. In such systems separation times of more than 50 minutes are required. The application of non porous stationary phases in HPLC generally enables a significant reduction in analysis time and solvent volume [14], to be achived.
The purpose of this study was to develop a HPLC procedure on non porous material specifically adjusted to the flavonoid complex of Filipéndula ulmaria L. (Maxim.). HPLC separation was combined with online mass spectrometric detection to ensure flavonoid detection in extracts without time consuming and expensive sample purification. To increase the sensitivity in quantitative MS-detection, a method in selected ion monitoring (SIM-mode) was developed. Thus, for the quality control of Filipendulae ulmariae flos and preparations from the drug, a new method for the fast identification and quantification of the flavonoids was elaborated and tested on commercial samples of meadowsweet flowers. The results obtained by HPLC/MS were compared to the results of spectrophotometry quantification.
EXPERIMENTAL
Material. Commercial samples of meadowsweet were purchased from Krauter- und Drogenhaus Kottas-Heldenberg&Sohn (Vienna, Austria) and Dr. Richter (Kufstein, Austria).
Acacetin, quercetin, kaempferol, hyperoside (querce-tin-3-O-galactoside), spiraeoside (quercetin-4'-O-gluco-side) and rutin (quercetin-3-O-rutinoside) were purchased from C. Roth KG (Karlsruhe, Germany). Aceto-
OH
OH
OH
HO
OH
"O-glucur OH O Quercetin-3-O-glucuronide
OH
HO
"O-arab OH O
Quercetin-3-O-arabinoside (Avicularosid)
HO
OH
O-gluc
OH OH O
Quercetin-4'-O-glucoside (Spiraeosid)
OH
OH
HO
OH
"O-galact OH O Quercetin-3-O-galactoside (Hyperosid)
HO ^
IL ^«L JL^
O-gluc-rham OH O
Quercetin-3-O-rhamnoglucoside (Rutin)
HO
O-gluc
OH OH O
Kämpferol-4'-O-glucoside
Fig. 1. Structures of the reported flavonoidglycosides from Filipendula ulmaria.
nitrile and methanol were HPLC grade from J.T. Baker (Deventer, Holland). Acetic acid of analytical grade was purchased from C. Roth KG (Karlsruhe, Germany).
HPLC Instrumentation, Columns and Conditions. HPLC separation was performed using a Perkin Elmer Series 200 liquid chromatograph connected to a PE Series 200 photodiode array detector and equipped with Turbochrom Navigator software. The analyses on the porous stationary phase were performed on Hyper-
Table 1. HPLC solvent gradient elution program employed for porous and non porous RP-HPLC separation of fla-vonoids in Filipendulae ulmariae flos
Porous Hypersil BDS® C-18 Non porous Kovasil™ MS C-18
time (min) Solvent composition (% mobile phase B) time (min) Solvent composition (% mobile phase B)
0 12 0 2
5 12 13 13
30 22.5 14 25
50 55 19 25
60 55 20 100
61 100 25 100
66 100 26 2
67 12 36 2
77 12
sil BDS® C-18 with 250 x 4.6 mm i.d. and 5 |m particle size (Hewlett Packard). Separation and quantification on the non-porous stationary phase were performed on a 33 x 4.6 mm i.d., 1.5 |m particle size, KovasilTMMS C-18 column (Chemie Uetikon AG, Switzerland). The solvents were (A) acetonitrile - 0.03 M acetic acid (2 + 98 v/v) and (B) acetonitrile - 0.03 M acetic acid (95 + 5 v/v). The separations were conducted at 25°C by multilinear gradient elution at a flow rate of 1 mL/min. UV-detection was set at 340 nm. The solvent gradient elution programms for both systems are listed in Table 1.
Mass Spectrometry. LC-MS analyses were performed with the HPLC system described above connected in series with a Perkin Elmer (PE) API-150 Sci-ex single quadrupole fitted with an ESI source. Processing of the data was performed with Analyst 1.1 software. Source and voltages were tuned on the molecular ion of rutin in a 10 |M solution of 80% methanol. The operating parameters in negative mode were set as follows: ionspray voltage -4200, temperature 300°C, declustering potential -1, focusing potential -100. For qualitative analysis full scan spectra in the range from m/z 200-650 amu (scan time 2 s) were recorded. In quantification scan spectra at specific molecular masses were taken with a bandwith of ±1 amu: m/z 283 for acacetin, m/z 285 for kaempferol, m/z 301 for quercetin, m/z 463 for spiraeoside and hyperoside and m/z 609 for rutin.
Standard and Sample preparation. From 80% (v/v) methanolic stock solutions of quercetin, kaempferol, hyperoside and rutin (each 1 mg/mL), spiraeosid (2 mg/mL), and acacetin (internal standard; 0.7 mg/mL)
standard solutions containing different amounts of the five flavonoids and 100 |L of the internal standard acacetin were prepared. The samples were extracted twice with 60% (v/v) methanol and evaporated to dryness. The extracts were dissolved in 3 mL 80% (v/v) methanol, 450 |L of these solutions were added to 50 |L of standard solution of acacetin.
Spectrophotometric analysis. Because of the similarities in the flavonoid patterns of meadowsweet flowers and birch leaves the determination of the total amount of flavonoids in meadowsweet samples was performend according to the method described in the monograph Betulae folium in the European Pharmacopoeia [15]. The spectophotometric quantification of the total flavonoid content in the samples was conducted on a Beckman DU 640 spectrophotometer at 425 nm.
RESULTS AND DISCUSSION
Qualitative Analysis. The most frequently used analytical technique for flavonoid separation is reversed phase high performance liquid chromatography (RPHPLC) on C-18 stationary phases [16]. To avoid peak-tailing in the separation of flavonoid glycosides an endcapped C-18 Hypersil column was chosen for method development. The best peak separation and sensitivity in mass spectrometric detection was obtained with a mobile phase of acetonitrile and 0.03 M acetic acid in double-linear gradient elution within 65 min. Identification of all main flavonoids in the samples was performed by LC-MS analysis and by comparing of the retention time of the peaks in the extracts with those of authentic reference substances. All known main flavonoids were detected exept quercetin-3-O-glucosid. In addition, traces of kaempferol were found, which has not previously been reported in meadowsweet flowers.
To reduce analysis time and necessary amounts of solvents, a method for sample separation on the non porous KovasilTMMS C-18 stationary phase was developed. The gradient profile was adapted to elute the main flavonoids and the internal standard acacetin within 20 min (Fig. 2). Though some components showed overlapping areas in UV-and TIC-profile, identification and separation with the Analyst software in MS-detection was easily possible in SIM mode.
Quantitative Analysis. In order to verify the applicability of the analytical method, a quantitative assay of fla-vonoids was carried out on two samples of Filipendulae ulmariae flos. The LC-MS system on KovasilTMMS C-18 was calibrated for hyperoside, rutin, spiraeoside, quer-cetin and kaempferol with four calibration points for each flavonoid (Table 2). Because of similarities in gly-cosidation and fragmentation behavior avicularoside was evaluated as hyperoside and kaempferol-4'-O-glu
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