Anal Bioanal Chem (2012) 403:2859–2867 DOI 10.1007/s00216-012-5800-6
ORIGINAL PAPER
DART-Orbitrap MS: a novel mass spectrometric approach for the identification of phenolic compounds in propolis Elena S. Chernetsova & Maciej Bromirski & Olaf Scheibner & Gertrud E. Morlock
Received: 28 November 2011 / Revised: 22 January 2012 / Accepted: 26 January 2012 / Published online: 23 February 2012 # Springer-Verlag 2012
Abstract This is the first direct analysis in real-time mass spectrometry (DART-MS) study of propolis and a first study on the analysis of bee products using high-resolution DARTMS (DART-HRMS). Identification of flavonoids and other phenolic compounds in propolis using direct analysis in realtime coupling with Orbitrap mass spectrometry (DART-Orbitrap MS) was performed in the negative ion mode for minimizing the matrix effects, while the positive ion mode was used for the confirmation of selected compounds. Possible elemental formulae were suggested for marker components. The duration of one sample analysis by DART-MS analysis lasted ca. 30 s, and all benefits of high-resolution mass spectrometry were used upon data processing using the coupling of DART with the Orbitrap mass spectrometer. The possibility Published in the special paper collection Recent Advances in Food Analysis with guest editors J. Hajslova, R. Krska, and M. Nielen. E. S. Chernetsova : G. E. Morlock Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany G. E. Morlock (*) Institute of Nutritional Science, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26, 35392 Giessen, Germany e-mail:
[email protected] e-mail:
[email protected] E. S. Chernetsova (*) People’s Friendship University of Russia, Miklukho-Maklaya st. 6, 117198 Moscow, Russia e-mail:
[email protected] M. Bromirski : O. Scheibner Thermo Fisher Scientific, Hanna-Kunath-Strasse 11, 28199 Bremen, Germany
for scanning analysis of dried propolis extract spots on a planar porous surface was investigated in the heated gas flow of the DART ion source with adjustable angle. As an independent method, the approach of scanning analysis is of high interest and of future potential for confirmation of the results obtained from liquid sample analysis. Scanning analysis is highly promising for further development in the bioanalytical field due to the convenience of the storage and transportation of dried sample spots. Keywords Direct analysis in real-time . DART-Orbitrap MS . Negative ion . Phenolic compounds . Propolis . Dried extract spots
Introduction Direct analysis in real-time mass spectrometry (DART-MS), reported for the first time by Cody et al. in 2005 [1], is a rapidly emerging method, which is highly promising in all fields of analysis of small organic molecules due to the minimization or even absence of the need for sample preparation. Several reviews have recently been made describing the developments in DART-MS, as well as its benefits and limitations [2-7]. In the first publications, the capabilities of DART-MS coupling were restricted to the mass resolution of 6,000 full width at half maximum (FWHM) of the time-of-flight (TOF) analyzer, because the DART ion source could only be used with the AccuTOF mass spectrometer manufactured by JEOL (Tokyo, Japan). The modern DART ion sources can be coupled with a wide range of different mass analyzers, therefore enabling the studies at the maximized reliability and value of information. A recent review of the literature available for DART-MS revealed more than 130 peer-review publications [2, 3], but in the majority of
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Fig. 1 Characteristic colored zone pattern for B- and O-types of propolis samples after separation by HPTLC [18]
them DART is still coupled to mass analyzers of low to medium resolving power such as quadrupole and TOF [7]. The coupling of DART to high-resolution mass spectrometry (HRMS) has been described only in a few studies for Orbitrap [8-14] and for a custom-built DART and Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) [15]. Therefore, further studies for such couplings for the demonstration of method capabilities and obtaining the maximal DART-MS performance are of interest. Due to the fast development of metabolomics and other interdisciplinary research in medical and nutritional sciences, novel methods for identification of naturally occurring components or other metabolites in biological samples and food are
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especially in demand. The first publications on such DARTHRMS studies are authored by Hajslova and co-workers and focus on multiple analysis of mycotoxins in cereals [10] and beer [11], and on the analysis of isoflavones in soybeans [14]. Bee product analysis by DART-MS is of particular interest due to its novelty and perspectives [3]. Only two DART-MS studies of bee product have been presented until now and both were investigations of honey: a presentation on determination of floral origin of honey by the principal component analysis of DART-MS TOF spectra [16], and a study on the possibility for the quantitation of 5-hydroxymethylfurfural in honey that shows the limitations of DART-MS due to the thermal degradation of sugar matrix into the analyte [17]. Propolis, a complex product collected by bees from resinated buds of different plants and converted in their organs, has been used in folk medicine for hundreds of years. Studying the composition of flavonoids and phenolic components in propolis with DART-Orbitrap MS coupling is of particular interest because of a growing interest for the use of propolis in medicine and cosmetics. Until now, no DART-MS studies of propolis have been performed. Therefore, the current study was aimed at the evaluation of capabilities of DART-Orbitrap MS coupling for identification of flavonoid and phenolic marker compounds, present in different samples of German propolis.
Experimental Reagents and chemicals All chemicals were of reagent-grade or better. Highperformance thin-layer chromatography (HPTLC) plates
Fig. 2 Characteristic DART mass spectra for B- and O-types of propolis samples (manual Dip-it introduction of liquid extracts)
DART-HRMS identification of phenolic compounds in propolis
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Table 1 Characteristic flavonoids and other phenolic compounds found in the negative ion DART mass spectra of propolis extracts
m/z exp.
Elemental formula, ion
RDB
δm, mass units
Elemental formula, compound
Propolis component
Number of citations in literature for presence in propolis
Structure
O
151.0393
C 8 H7 O 3
5.5
-1.2×10
-4
C 8 H8 O 3
vanillin
O
91
OH H
163.0393
C 9 H7 O 3
6.5
-1.3×10
-4
C 9 H8 O 3
coumaric acid
HO
OH
211
O HO
179.0343
C 9 H7 O 4
6.5
-1.1×10
-4
C 9 H8 O 4
caffeic acid
HO
OH
263
O O
ferulic acid 193.0501
C 10 H9 O 4
6.5
0.7×10
-4
HO
OH
203
O
C 10 H10 O 4
HO
methyl caffeate
HO
O
11
O HO
247.0973
C 14 H15 O 4
7.5
2.8×10
-4
C 14 H16 O 4
prenyl caffeate
HO
O
33
O
HO
253.0508
C 15 H9 O 4
11.5
7.2×10
-4
C 15 H10 O 4
O
chrysin
327 O
HO
253.0868
C 16 H13 O 3
10.5
3.7×10
-4
C 16 H14 O 3
O
methoxy flavanone isomers
18 O O e. g., 6-methoxyflavanone
HO
O
pinocembrin 255.0661
C 15 H11 O 4
10.5
4.4×10
-4
253
C 15 H12 O 4
O
HO
OH
isoliquiritigenin (or liquiritigenin)
OH
HO
13 HO
O
HO
benzyl caffeate
HO
O
42
O
269.0820
C 16 H13 O 4
10.5
1.1×10
-3
C 16 H14 O 4 O
O
pinostrobin
74 HO
O
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Table 1 (continued)
m/z exp.
Elemental formula, ion
RDB
δm, mass units
Elemental formula, compound
Propolis component
Number of citations in literature for presence in propolis
Structure
OH HO
O
95
naringenin
271.0611
C 15 H11 O 5
10.5
4.4×10
-4
HO
O
C 15 H12 O 5 HO
O
pinobanksin
137 OH HO
283.0974
C 17 H15 O 4
10.5
3.8×10
-4
C 17 H16 O 4
caffeic acid phenethyl ester (CAPE)
O
HO HO
393
O O
O HO
O
ermanin
13 O HO
313.0716
C 17 H13 O 6
11.5
4.3×10
-4
O
C 17 H14 O 6 HO
O
pinobanksin 3-acetate
44 O HO
O O
silica gel 60 (20×10 cm, layer thickness ca. 200 μm, Merck, Darmstadt, Germany) were used. A set of HPTLC plates was prewashed with a mixture of methanol and water (7:1, v/v) and dried in an oven at 110 °C for 20 min. The prewashed plates (covered by a glass plate and wrapped in aluminum foil) were stored in a desiccator until usage. The first plate type was used for experiments with dried propolis spots (DPS).
tracks of 6 mm), and distance from lower edge 8 mm. The digital documentation was performed in the reflectance mode at white light illumination using the DigiStore 2 Documentation System (CAMAG) consisting of illuminator Reprostar 3 with digital camera Baumer optronic DXA252. Data were processed with winCATS software, version 1.4.5 (CAMAG). DART-MS
Samples Propolis extracts were obtained from the Apicultural State Institute (Stuttgart, Germany) [18]. The extracts were analyzed by DART-Orbitrap MS coupling directly or as DPS on an HPTLC plate. The application of the propolis extracts onto HPTLC plates was performed bandwise using the Automatic TLC Sampler 4 ATS4 (CAMAG, Muttenz, Switzerland). The volumes applied were 20 μL. The following application pattern was used for DPS resulting in 18 tracks per plate side: band length 4.5 mm, track distance 10.5 mm (i.e., a distance between
The “DART-SVPA” ion source (IonSense, Saugus, MA, USA), equipped with a motorized linear rail, was coupled to the Thermo Scientific Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via the Vapur vacuum interface (IonSense). The evacuation was performed using a Diaphragm Vacuum Pump MZ 2 (Vacuubrand, Wertheim, Germany). The DART ion source was operated using the following conditions: negative or positive ionization mode; temperature of 250 °C. The grid voltage was +350 V in the positive mode and −350 V in
DART-HRMS identification of phenolic compounds in propolis
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Fig. 3 Signal of the chrysin [M−H]− ion in the DART negative ion mass spectrum of the B-type propolis sample with the abundant signal of methoxyflavanone (A) and further confirmation of chrysin and methoxyflavanone in propolis extract by their [M+H]+ ions in the DART positive ion mass spectra (B)
the negative mode. For operation, helium gas (99.999%) was employed, whereas nitrogen gas (99.999%) was used in the standby mode. For DART operation, the DART-SVP web-based software (IonSense) was used. For data acquisition and processing, Xcalibur software (Thermo Fisher Scientific) was used. The data were collected in the m/z range of 70–700 at the speed of two scans per second, providing the resolution of 50,000 (FWHM) at m/z 200. Analysis of liquid samples was performed using the DART ion source in a horizontal alignment. The extracts were manually introduced into the ionization region using glass Dip-it sampling tips (IonSense). The analysis of DPS on HPTLC plates was performed using the DART angle of 30° and supplying the HPTLC plate into the ionization region with a motorized rail (IonSense) at the speed of 0.2 mm s−1 on a self-built stainless steel table [19].
Results and discussion Analysis of liquid propolis extracts In a recent HPTLC study [18], the German propolis samples were classified into two main types, according to the characteristic pattern of flavonoid or other phenolic marker compounds visible on the HPTLC plates after selective derivatization. Exemplarily, four different samples are illustrated and samples showing the bluish pattern were marked as B-type, while the samples with a dominating orange color were marked as O-type (Fig. 1, [18]). The identification of unknown flavonoids and other phenolic compounds in these two types of propolis samples was of interest. Thus, the capabilities of coupling DART with the Orbitrap mass spectrometer were studied to identify marker components of the
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two propolis sample types. Initially, the direct analysis of the propolis extracts was performed using DART-MS and introducing the extracts on the glass Dip-it tips manually into the ionization region. Using the negative DART ionization mode, flavonoids and phenolic compounds were easily ionized forming [M−H]− ions, while the ionization of other extract components was minimal. Hence, due to the acidic nature of flavonoids and phenolic compounds, their signals were dominant in the DART mass spectra of the propolis extracts. The interference with other extract components was rated as low (Fig. 2). The high resolution of the mass analyzer enabled the determination of the m/z values for the characteristic ions in the mass spectra with an accuracy of better than 2 ppm. Using the software, the possible elemental formulae were easily calculated for the ions of interest from the DART mass spectra. As it followed from the preliminary studies of flavonoids and phenolic compounds, they preferably form the [M−H]− ions in the negative DART-MS mode; therefore, the elemental formulae could be suggested for the respective extract components from their m/z values. The RDB (ringplus-double-bonds) values, calculated with Xcalibur software, were used in the range of 4–12 as a filter to find the compounds with possible flavonoid or phenolic structures. After composing a list of possible elemental formulae for marker compounds, these formulae were subjected to extensive search by means of the SciFinder scientific search engine, to find the possible structures and to check if such compounds have been previously reported as propolis components in the literature. According to this search, the respective signals of mass spectra in Fig. 2 could belong to the flavonoids and phenolic compounds listed in Table 1. The average mass difference between the theoretical and experimental formulae for the listed compounds was 0.3×10−3 mass units or ca. 1 ppm, which was within the accuracy of the method. Table 2 Confirmation of the molecular formulae for characteristic flavonoids and phenolic compounds by their signals in the positive ion DART mass spectra
Within the current study, the data obtained previously for flavonoid and phenolic marker compounds in propolis extracts using HPTLC [18] and HPTLC-ESI-MS [20], were generally confirmed with DART-Orbitrap MS, but the time per sample analysis in case of DART-MS was minimized down to ca. 30 s per sample (sample introduction 5 s plus waiting time between sample introductions 30 s), while HPTLC analysis of 24 samples on a plate took 30 minutes and required further operations on post-chromatographic derivatization or coupling with ESI-MS. In the HPTLC studies chrysin has been identified as one of the markers of interest in the propolis extracts. The abundant signal at m/z 253.0868, observed in the negative ion DART mass spectra for the B-type samples, could not be assigned as chrysin, as the theoretical m/z value of the chrysin [M−H] − ion was 253.0501, but in the zoomed m/z range between 252.4 and 253.6 the chrysin signal was easily recognized with a mass accuracy of 0.7×10−3 mass units (Fig. 3A). This clearly shows the advantage of the high resolution of the Orbitrap mass analyzer at the presence of a highly abundant ion at the same integral-valued m/z value as the analyte. And finally, the abundant signal at m/z 253.0868 was assigned as methoxyflavanone isomer (Table 1). Using the positive DART ionization mode, the mass spectra for propolis extracts were more complex due to the simultaneous intensive ionization of other classes of extract components, because in the positive mode the majority of classes of organic compounds are easily ionized by DART [2]. However, when necessary, positive ion DART mass spectra could be used as an additional tool for the confirmation of the presence of flavonoids and phenolic compounds by the m/z values of their [M+H]+ ions, determined with the high accuracy of the Orbitrap mass analyzer. For example, the enlarged region of the positive ion DART mass spectrum of the B-type sample (Fig. 3B) confirms the simultaneous presence of chrysin and the methoxyflavanone
Propolis component
m/zexp.
Ion
Elemental formula, compound
δm, mass units
Vanillin Coumaric acid
153.0544 165.0542
[M+H]+ [M+H]+
C8H8O3 C9H8O3
−7.4×10−4 −9.4×10−4
Caffeic acid Ferulic acid/methyl caffeate Prenyl caffeate Chrysin Methoxyflavanone isomers Pinocembrin/isoliquiritigenin/liquiritigenin Benzyl caffeate/pinostrobin Naringenin/pinobanksin Caffeic acid phenethyl ester (CAPE) Ermanin/pinobanksin-3-acetate
181.0494 195.0648 249.1116 255.0646 255.1006 257.0803 271.0955 273.0750 285.1110 315.0852
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
C9H8O4 C10H10O4 C14H16O4 C15H10O4 C16H14O3 C15H12O4 C16H14O4 C15H12O5 C17H16O4 C17H14O6
−6.7×10−4 −9.3×10−4 −1.1×10−3 −1.2×10−3 −1.5×10−3 −1.1×10−3 −1.5×10−3 −1.3×10−3 −1.7×10−3 −1.7×10−3
DART-HRMS identification of phenolic compounds in propolis
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Fig. 4 DART chronogram and a mass spectrum for a B-type propolis sample after scanning and recording the respective dried propolis spots (DPS) on an HPTLC plate. Automated introduction of the HPTLC strip
into the ionization region was performed using a motorized linear rail at a speed of 0.2 mm/s
isomer in a sample by the respective [M+H]+ ions. For all the flavonoid and phenolic compounds identified in the negative ion DART mass spectra, the respective [M+H]+ ions were found in positive ion spectra, effectively confirming their molecular formulae (Table 2). In a quantitative DART-Orbitrap MS study of isoflavones in soybeans [14], the higher abundance of isoflavone ions in the negative ion mode was reported for model compounds, while only positive ionization was used for the real sample studies, because the internal standard compound was not ionizable in negative mode. As it follows from the current study, in qualitative studies of the composition of phenolic compounds in biological samples using DART-Orbitrap MS, the identification will be more effective and reliable if using the current approach, when negative ion spectra are used for identification of phenolic marker compounds, and positive ions are
used for the confirmation of the presence of respective compounds in samples. Analysis of dried propolis spots (DPS) Another interesting potential field for DART-MS studies is analysis of dried spots of liquids on a carrier. Such analysis can be useful in cases when the storage of samples in their liquid state is inconvenient, e.g., due to restrictions with regard to transportation or storage. The approach for MS analysis of dried liquids on a carrier surface was demonstrated in 2009 for dried blood spot (DBS) analysis using a thin-layer chromatography mass spectrometer interface and ESI ionization [21]. The impressive development on DBS analysis using ESI-MS since the first publication on DBS analysis in 2009 [21] confirms the especial promise of
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DART-MS in the bioanalytical field, because analysis in the flow of gas from the DART source is faster than ESI-MS analysis and does not involve the use of any solvents for spot extraction, which can influence its composition due to impurities contained in such solvents. In 2010, the horizontally aligned DART ion source coupled to a FTICR-MS was used for metabolic profiling of lipids in human breast milk applied as spots on paper, which was reported at a conference [22]. However, in respect of DART-MS analysis, paper may not be considered as a perspective carrier due to its easy deformation and low stability under heating. The thirdgeneration DART ion source “DART-SVPA” is capable of being set off axis to the sampling surface for direct surface analyses, giving rise to the studies in scanning surface analysis using DART-MS. Using such a source, the capability of quantitation of several drugs from DBS on glass slides has been proven using a triple quadrupole mass spectrometer in a pharmacokinetic study [23]. To our knowledge, no other studies on DART-MS analysis of dried spots on a carrier have been performed until now. In the current study, the opportunity to analyze dried propolis spots (DPS) on an HPTLC plate used as a porous carrier instead of analyzing liquid samples was studied using DART-Orbitrap MS. For such a coupling, the HPTLC plate strip, containing the spots of interest, was fixed on a selfbuilt stainless steel carrier table [19]. The DART-MS scanning of such a strip in negative ion mode was automatically performed at a speed of 0.2 mm/s using the motorized linear rail of the DART source. DPS volumes of 20 μL for seven different propolis extracts were applied on an HPTLC plate as bands of 4.5 mm. Then they were automatically scanned along (Fig. 4). The recording was performed at an increased Fig. 5 The DART mass spectrum of the background from the HPTLC plate
E.S. Chernetsova et al.
DART temperature of 350 °C to provide an improved desorption of propolis extract components from the HPTLC plate. Due to this, the pattern of the DART mass spectra differed from the pattern of spectra obtained from liquid solutions. Signals of volatile components in the low mass range were more abundant, e.g., the abundant signal at m/z 121.0286 on the background-corrected mass spectrum could belong to [M−H]− ion of benzoic acid. However, all characteristic signals for B-type propolis samples (e.g., C8H7O3, C9H7O3, C10H9O4, C16H13O3) were also registered in this spectrum. The background correction was performed by subtracting the mass signals obtained from a blank region (Fig. 5) from the original substance spectrum at the same HPTLC plate. By doing so, any system peaks can be mitigated in a substance spectrum. This confirmed the general applicability of such an approach utilizing the scanning analysis of dried extracts on an HPTLC or TLC carrier (so-called spots) for analysis. Besides, it confirms that, along with the storage/transportation considerations, analysis of DPS can also be helpful when used as an additional tool for the confirmation of results of direct liquid extract analysis.
Conclusions The effective combination of DART with an Orbitrap mass spectrometer as a powerful small-molecule identification tool has been proven for the identification of flavonoids and other phenolic compounds in propolis extracts, either in liquid solution or analyzed as DPS on a porous carrier surface (HPTLC plate). The high mass resolution, provided with such
DART-HRMS identification of phenolic compounds in propolis
a coupling, was used to find the possible elemental formulae for marker components. The matrix effects were minimized using the negative DART ionization mode, in which such acidic analytes as flavonoids and phenolic compounds were easily ionized forming abundant [M−H]− ions. The positive ion DART mass spectra proved to be effective tools for molecular formulae confirmation of the analytes of interest. The duration of one sample analysis by DART-MS lasted ca. 30 s, and all benefits of high-resolution mass spectrometry were used upon data processing using the coupling of DART with the Orbitrap mass spectrometer. Switching between DART ionization modes, as well as changing the ion sources (DART, ESI) can easily be performed within seconds and several minutes, respectively. ESI ionization can serve as a confirmative tool for the presence of specific ions. The possibility of analysis of dried spots of liquids on a carrier using DART-MS, reported only in two studies [22, 23] for spots of milk on paper and for DBS on glass slides, has been confirmed using an HPTLC plate as a porous carrier and the DPS scanning analysis by the DART ion source with an adjustable angle. This gives rise for further studies of dried spots on a flat carrier by DART-MS—used for the confirmation of liquid sample analysis or as an independent analytical method. Due to the convenience of the storage and transportation of dried spots, this approach is highly promising for further development, especially for many other bioanalytical applications, e.g., metabolomics studies. Acknowledgments The authors are grateful to Elizabeth Crawford and Dr. Brian Musselman (both IonSense, Saugus, MA, USA), Sue Kennerley and Pete Ryan (both KR Analytical Ltd., Sandbach, UK) for technical support and fruitful discussions, to Nadine Kunz and Dr. Annette Schroeder (both Apicultural State Institute, Stuttgart, Germany) for collecting the propolis samples and preparation of the propolis extracts, to Irina Scholl for the HPTLC fingerprint screening (supervised by G. Morlock during her diploma thesis), to WALA Heilmittel GmbH for initiating and financing the preliminary project, and to Professor Dr. Wolfgang Schwack (University of Hohenheim, Stuttgart, Germany) for his valuable assistance getting in contact with Thermo Fisher Scientific and the excellent research conditions at the Institute of Food Chemistry. This work was financially supported within the program Erasmus Mundus Action 2 “IAMONET-RU”.
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