WO1999057748A1 - Method for analyzing trans-resveratrol by laser desorption coupled to resonant multiphotonic ionization - Google Patents

Abstract

The invention relates to a method for analyzing trans-resveratrol by laser desorption associated to resonant multiphotonic ionization, said method comprising the three steps: laser desorption of the molecules; resonant multiphotonic ionization of the desorbed molecules; detection of the ions through mass spectrometry per flight-time. The ionization must be carried out in synchronization with the desorption and to resonant wave length of said compound, that is between 302 and 307.5 nm. The present invention can be applied to the analysis of trans-resveratrol both in natural samples and in manufactured products, whether solid or liquid. The various techniques used up to now for the trans-resveratrol analysis require long and complex operations for preparing the sample, making the analysis method difficult and more expensive, these techniques being also the main sources of errors. The main innovation of the present method based on a direct analysis which increases the accuracy and precision of the measurements thereby facilitating considerably the analysis method.

Description

TITLE.

Analysis method of trans-resveratrol by laser desorption coupled to resonant multiphoton ionization

OBJECT OF THE INVENTION

The present invention relates to a method of direct analysis of trans-resveratrol (3, 5, 4'-trihydroxystilbene) in both natural samples and products manufactured by a machine that combines laser desorption with subsequent resonant multiphoton ionization using a second laser. and detection by mass spectrometry by time of flight Ionization must be performed at the resonant wavelength of this compound, which we have determined to occur in the wavelength range between 302 and 307 5 nm

The present invention falls within the field of the application of laser technology to the chemical analysis of food

BACKGROUND.

In the bibliography, you can find various methods for the analysis of resveratrol, especially in wines, due to the enological interest of this compound. In general, all of them are chromatographic methods, both by gas chromatography (CG) and high performance liquid chromatography (HPLC). Before the chromatographic resolution of the compound, it is necessary to carry out various operations for the separation and extraction of the compound, the analytical techniques used are: extraction with organic solvents (see for example; P. Jeandet et al. J. Agrie. Food Chem. 43 (1995) 316; EH Siemann et al Am. J. Enol. Vitw. 43 (1992) 49; O Lamikanra et al J. Agrie. Food Chem. 44 (1996) 1111), solid phase extraction (D M. Goldberg et al Anal. Chem. 66 (1994) 3959 and Am. J. Enol. Vitic. 46 (1995) 159) and direct injection (GJ Soleas et al Anal. Chem. 69 (1997) 4405, JP Roggero et al. S Ali ents 15 (1995) 411) Also in the methods that use chromatogr In order to obtain gases, the derivation is generally necessary with bis- [trimethylsylil] -trifluoroacetamide, (GJ Soleas et al Am. J. Enol. Vitπ. 46 (1995) 346, EN Frankel et al J. Agrie. Food Chem 43 (1995) 2

890), with subsequent detection by flame ionization or mass spectrometry. In the HPLC methods the detection used is based on ultraviolet absorption (A. Gonzalo et al. J. Wine Res. 6 (1995) 213; DM Goldberg et al. J. Chromatogr. A 708 (1995) 89), fluorescence (R. Pezet et al. J. Chromatogr. A 663 (1994) 191), electrochemistry (KD McMurtrey et al. J. Agrie. Food Chem 42 (1994) 2077) or diode network (F. Mattivi et al. J . Agrie. Food Chem 43 (1995) 1820; DM Goldberg et al. Anal. Chem. 68 (1996) 1688; RM Lamuela Raventós et al. J. Agrie. Food Chem. 43 (1995) 281.

Although no inter-laboratory comparison has been performed, it is tacitly assumed that any of these methods is correct and would lead to the same results in the analysis of a given sample, however it has been observed that in some cases different researchers they obtain concentrations that differ by up to an order of magnitude for the same type of wine. A study has recently been published in which a comparative evaluation of four of these methods of analysis of resveratrol in wine is carried out and the values published by other authors are reviewed (GJ Soleas et al. Am. J. Enol. Vitic. 48 ( 1997) 169). The great variability found is mainly attributed to the possibility of isomerization during the derivation processes, significant losses during the multiple extraction stages, and the presence of various resveratrol-derived compounds that may interfere with the results. In any case, it is evident that the different techniques used to date for the analysis of trans-resverarol in wine require long and complex sample preparation operations prior to analysis. These operations not only hinder and make the analysis process more expensive, but are one of the main sources of error. With the analysis method claimed in the present patent application we consider these problems are solved.

DESCRIPTION OF THE INVENTION.

The present invention relates to a method for direct analysis of trans-resveratrol by combining laser desorption with subsequent resonant multiphoton ionization of the desorbed molecules and subsequent detection by time-of-flight mass spectrometry. The main innovation of our method is the realization of a direct analysis, since it does not require a previous treatment of sample preparation or separation as in the case of chromatography. This allows increasing the accuracy and precision of the technique by eliminating one of the main sources of error present in most of the techniques that require this type of treatment. The absence of previous treatment supposes, in addition, a great saving of time and economic cost, facilitating the analysis, reducing the consumption of solvents, etc. In addition, as seen in the previous section, it is in this pre-treatment of the sample that the main problems presented by the analysis of the trans-resveratrol content using the techniques currently used appear.

The experimental setup, (Figure 1), is a time-of-flight mass spectrometer coupled to a laser desorption and ionization chamber. Said system basically consists of three parts: vacuum equipment, lasers and electronic equipment. The high vacuum equipment consists of two chambers: in the first one the desorption of the sample to be analyzed is performed, the ionization of the desorbed molecules and the acceleration of the ions by means of plates connected to three high voltage sources, each acquiring a speed different depending on its mass. Subsequently, the ions enter the second chamber, which is a flight time tube of approximately lm in length, where the electric field is null; this allows the ions to be separated due to their different speed and therefore, according to their mass. The experimental system uses two high energy pulsed lasers; one of them, the one used for ionization, can be tuned to choose the appropriate wavelength for each molecule of analytical interest. Detection is carried out by a system of microchannel dishes located at the end of said chamber. The obtained signal is digitized in an oscilloscope coupled to a computer; This signal consists of a flight time spectrum in which each peak corresponds to a mass. Figure 2 shows the internal parts of the experimental system in more detail, as well as the interaction and direction between both lasers. The sample is prepared as explained in the section entitled "Preferred embodiment" and is deposited on a pyrex disc using an airbrush. The disc is located parallel to the first accelerator plate approximately 2 mm from it; during desorption the disc is rotated by a stepper motor and in turn is Located on a positioner that allows the direction perpendicular to the desorption laser to be varied, which allows to always have a fresh surface on which to desorb. For desorption, the second harmonic of a Nd: YAG laser is used, which passes through the disc at its rear and desorbs the sample; the desorbed molecules expand in the region of acceleration by the action of the vacuum, and are ionized by the second laser pulse perpendicular to the first.

Our invention relates not only to the tuning of the method, but to the wavelength range (302 to 307.5 nm) where the resonant ionization of the trans-resveratrol molecule occurs. This non-linear process is possible thanks to the high intensity of laser radiation. A multiphoton absorption process can in principle pass through real or virtual levels of the molecule: the half-life of a virtual level is around 10 ^"15 seconds, while that of a real level is around 10 ^"9 and 10 ^{" 6} seconds, so the probability of absorption of more than one photon is greater if it is done through a real state. This process is called resonant multiphoton ionization and is generally known by its acronym in English: REMPI, {Resonance Enhanced Multi-Photon lonizatiori).

Trans-resveratrol has been shown to undergo a biphotonic ionization process, the specific example of which is shown in figure 3, which shows the bilogarithmic representation of the intensity of the trans-resveratrol signal versus ionization energy, keeping the rest of the experimental conditions (desorption energy, accelerating voltages, etc.) constant. According to the multiphoton absorption formulas, the slope of this representation indicates the number of photons absorbed by the molecule in the ionization process; In the case of trans-resveratrol, the experimental data fit a straight line with a slope of 1.9 ± 0.1, which is consistent with a biphotonic resonant process (R2PI) with a single color.

The main advantage of the REMPI technique is the selective ionization of a certain molecule in a complex mixture: obviously the electronic states are different for each molecule, even when they are structurally similar. By varying the wavelength of the laser, it is possible to determine the wavelength that is resonant with the compound of interest, but not with the rest of the compounds present in the sample. Taking into account that to produce a REMPI process much less irradiance is necessary than for non-resonant ionization processes, selecting the length of Adequate wave and decreasing the energy of the laser, it is possible to obtain a simple spectrum that contains only the peak of the molecular ion to be analyzed. By this technique it is therefore possible to eliminate the matrix effects present in most conventional analytical techniques. One of the necessary requirements for the analysis using Resonant Ionization Mass Spectrometry is therefore to know the REMPI spectrum of the analyte, in order to be able to select the optimal wavelength for the selective ionization of the compound. According to the studies we have carried out, the wavelength range in which trans-resveratrol is resonant is from 302 to 307.5 nm, with the maximum absorption at 302.1 nm as can be seen in Figure 4.

BRIEF DESCRIPTION OF THE FIGURES Figure 1:

Figure 1 shows a schematic view of the experimental setup, comprising both the high vacuum chambers, the lasers and part of the necessary electronics. The abbreviations used are the following:

CI: Ionization chamber.

TOF: Flight time tube. G: Knife Gate Valve.

Ll: Nd: YAG lasers used for desorption.

L2: Dye Laser used for ionization.

US: Pulse generator for synchronization between both lasers.

VI -V3: High voltage sources, connected to their respective accelerator plates. V4: Detector supply voltage.

D: Detector of (2) microchannel plates.

Ose: Digital Oscilloscope.

PC: Personal computer.

Figure 2:

Figure 2 shows the internal parts of the ionization chamber in more detail, mainly: Ll, L2: Laser systems, as indicated above. Q: Pyrex disc. M: Sample to analyze. VI: First accelerator plate, ("repel"). V2: Second accelerator plate. V3: Einzel-type ionic lens. DI, D2: Deflectors in the directions perpendicular to the ion beam.

Figure 3: Figure 3 shows the bilogarithmic representation of the intensity of the trans-resveratrol signal, (In), versus the ionization energy, (E¡), maintaining the rest of the experimental conditions (desorption energy, voltages constant accelerators, etc.).

Figure 4: Figure 4 shows the REMPI absorption spectrum of trans-resveratrol in the range between 294 and 306 nm.

PREFERRED FORM OF EMBODIMENT The present invention is illustrated by the following examples, which are in no way limiting its scope, which is exclusively defined by the claim.

1.- Sample preparation. The preparation depends first of all on the state of the sample to be analyzed:

Example 1: In the case of liquid samples such as wine, juices, etc. This is deposited on a pyrex disc, by means of an airbrush that pulverizes the sample allowing the subsequent evaporation of the most volatile compounds, a thin and homogeneous film on the surface of the pyrex. The homogenization system consists of a motor that rotates the Pyrex plate at a constant speed. In front of the plate and at an adjustable distance is an airbrush that works continuously driving the sample by air at a variable pressure. The distance between the plate and the airbrush, the opening of the airbrush nozzle and the gas pressure must be optimized in each case, depending on the characteristics of the liquid to be deposited: viscosity, volatility, etc. If the liquid is not too volatile, as in the case of wine, it is necessary that the deposition is done as slowly as possible, to allow the liquid to evaporate on the surface of the disk. If necessary, a suitable solvent (acetone, methanol, ethanol, ...) could be used as a vehicle to facilitate evaporation.

Example 2: In the event that the sample is in this solid state (fruits, vegetables, etc.), it is crushed and dispersed in a suitable solvent that is used as a vehicle. For this, a disperser-homogenizer is used that ensures a particle size of 5 to 25 μm; Since the airbrush nozzle used is 0.4 mm in diameter, this particle size is sufficient to be able to carry out the deposition on the pyrex plate according to the procedure previously used.

2.- Desorption and ionization by laser.

Once the pyrex plate has been introduced into the ionization chamber, according to the scheme shown in Figure 2, the molecules are desorbed using the second harmonic of a Neodymium-Y AG laser that is introduced into the chamber without focusing. The flow of the desorption laser must be optimized so that the maximum signal is obtained, while avoiding the fragmentation of the desorbed molecules. The desorbed molecules are subsequently ionized by a second laser pulse. In this case, a tunable laser must be used, since the working wavelength depends, as seen above, on the molecule to be ionized. In the case of resveratrol, we have determined that the resonant wavelength range is 302 and 307.5 nm, with the maximum absorption being approximately 302.1 nm; this must therefore be the optimal working wavelength to achieve selective trans-resveratrol ionization in complex samples. The following table lists other important experimental parameters used in the analysis of trans-resveratrol. Pressure in the ionization chamber 1x10 ^' "- 5xl0 ^{' v}

Pressure in TOF chamber 1x10 ^" '- 5x10 ^" *

Vi (volts) 1540

V ₂ (volts) 1350

V ₃ (volts) 230

V (volts) 2000

Desorption-ionization distance (mm) 7.5

Frequency of both lasers (Hz) 10

Laser pulse duration (nm) 4 - 6

Delay between both lasers (μs) 25 λ Desorption (nm) 532 λlonization (m) 302.1

Edesorption (mJ / pulsθ) 8

Elαniacrin (μJ / pulS0) 83

Table 1-. Experimental system conditions

Claims

1 - Method for trans-resveratrol analysis by laser desorption coupled to resonant multifotonic ionization, characterized in that it uses two high-vacuum chambers, two pulsed high-energy lasers and a mass-spectrometry detection system per flight time. 2 - Method for the analysis of trans-resveratrol by laser desorption coupled to resonant multifotonic ionization, according to claim 1, characterized in that it consists of two stages: - a desorption stage an ionization stage performed synchronously in the first high vacuum chamber. 3. Method for the analysis of trans-resveratrol by laser desorption coupled to resonant multifotonic ionization, according to claim 1, characterized by performing the ionization of the compound in the wavelength range in which it is resonant, between 302 and 307, 5 nm and preferably at the maximum absorption which is 302.1 nm.