EP1481416B1 - Mass spectrometry method for analysing mixtures of substances - Google Patents

Description

The present invention relates to a mass spectrometric method for the analysis of substance mixtures with a triple quadrupole mass spectrometer.

In the analysis of complex mixtures of substances of biological and / or chemical origin, the analyst, in addition to the task of identifying the structure of individual substances contained in the mixture, repeatedly encounters the problem of detecting and possibly quantifying all substances present in the mixture. This should be done as quickly as possible and with a high degree of accuracy, ie with a small error deviation. This becomes all the more important when information about a biological system is to be obtained, for example, from a microorganism grown under certain fermentation conditions or from a plant grown under different environmental conditions or from a wild-type organism such as a microorganism or a plant compared to its genetically altered mutant , Such comparisons are required to allow assignment of mutations of unknown genes in the genome of these organisms to a particular metabolic phenotype.

The success in the analysis of these mixtures of substances, for example chemical synthesis approaches from combinatorial chemistry or from extracts of microorganisms, plants or parts of plants depends to a great extent on the speed and reproducibility of the analytics used. In such a screening, a large number of samples must be screened, therefore, fast, simple, highly sensitive and highly specific analytical methods are required.

A major problem of this analysis is the rapid, simple, reproducible and quantifiable identification of the substances contained in the mixtures. As a rule, separation methods such as thin-layer chromatography (= TLC), high-performance liquid chromatography (= HPLC) or gas chromatography (= GC) are used to analyze the products. However, with the help of these chromatographic methods, a broad range of substances can not be quickly and easily identified and quantified. Also methods such as NMR or mass spectrometry are described for this task. However, as a rule, some preparation of the samples is required for these analysis methods, such as work-up over, for example Salt precipitation and / or subsequent chromatography, concentration, desalting of the samples, buffer exchange or removal of any detergents contained in the sample. After this pretreatment, the samples can be used for the aforementioned analyzes and individual substances in selected samples can be identified and quantified. However, these methods are time-consuming and allow only a limited sample throughput, so that such analysis methods in so-called high-throughput screening (= HTS) or the broad screening of substance mixtures in biological or chemical samples do not apply. An advantage of very precise methods, such as NMR or IR spectroscopy, is that they provide information both about the structure and optionally about the quantity of a substance.

In order to enable a higher sample throughput in HTS, indirect, easily measurable methods such as color reactions in the visible range, turbidity measurements, fluorescence, conductivity measurements etc. are often used. These are in principle very sensitive, but also prone to failure. The disadvantage here is, above all, that many false-positive samples are analyzed in this procedure and, since they are indirect detection methods, there is no information about the structure and / or the quantity of a compound. In order to be able to exclude these false positives in the further course of action, further analytical methods are generally used after a first rapid analysis such as, for example, NMR, IR, HPLC / MS or GC / MS. This is again very time consuming.

Generally, it can be said that improving the sensitivity and predictive power of the detection methods results in a slowdown in the speed of analysis.

When working with complex biological mixtures such as extracts of microorganisms, plants and / or animals is also to be noted that individual compounds are present in the mixtures only in very small amounts or only small amounts of each sample itself for analysis available stand so that the method used must have a high sensitivity. Furthermore, for some analysis methods, the non-volatile buffers and / or salts often present in biological samples pose a problem, as they adversely affect the sensitivity of the methods or their use. The same applies to the presence of detergents in these samples.

For the analysis of complex sample mixtures mass spectrometric methods are known from the prior art, for example, from the Analysis of synthetic, petrochemical, environmental and biological samples. However, these methods are only used for the analysis of individual known compounds in these samples. Broad measurement series, for example in the context of an HTS or in the identification and quantification of a variety of compounds in these samples are not described.

Application is the coupling of gas chromatography and mass spectrometry (= GC / MS) for substances that are extractable from the mixtures and volatile. For the analysis of substances or analytes that can not be converted into the gas phase easily or with difficulty and in which a large excess of solvent present must be removed, the so-called liquid chromatography or high-pressure liquid chromatography -Mass Spectrometry (= HPLC / MS) used. An overview of the different LC / MS methods and their equipment is published by Niessen et al. (Journal of Chromatography A, 703, 1995: 37-57 ) refer to. In the US writings US 4,540,884 and US 5,397,894 Mass spectrometers and their structure are described and claimed.

With the aid of the abovementioned methods, it is possible to determine substances in a molecular weight range of up to 100 KD (= kilodalton), that is to say a broad range of substances, for example in a lower mass range of up to about 5000 D (= daltons), such as fatty acids, amino acids , Carboxylic acids, oligosaccharides or polysaccharides, steroids etc. and / or in a higher mass range above 5000 D, such as peptides, proteins, oligonucleotides and oligosaccharides or other polymers. High molecular weight materials such as coal tar, humic acid, fulvic acid or kerogen can also be analyzed ( Zenobie and Bone Must, Mass Spec. Rev., 1998, 17, 337-366 ). Both the identity and the structure of substances can be determined, although the structural analysis is not always clear, so it must be confirmed by other methods, for example NMR.

ELEsmans et al. (J. Chromatogr. A 794, 109-127 (1998 ) discloses in particular LC-MS methods for the analysis of nucleobases, nucleosides, nucleotides, oligonucleotides and DNA.

By G. Hopfgartner and F. Vilbois (Analusis, 2001, 28, No. 10, 906-914 ) describes a method for screening by means of LC / MS of in vitro or in vivo derived metabolites of structurally known compounds which are active agents in different phases of drug development. This procedure is done in two steps. In the first search step are in a rapid "full scan mode" captures interesting ions that are candidates for further investigation. These may be ions which correspond to ions of particularly high intensity or are candidates for possible degradation products or metabolites of the active substances. These ions are used in a second scan to identify the chemical structure of these ions or compounds after fragmentation in a collision chamber of the mass spectrometer. In order to enable a rapid elucidation of the ion or metabolite structure, the collision chamber constantly contains collision gas. A disadvantage of the structure determination is that a known mass of a precursor ion, a fragment or an ion adduct is required. Advantageously, the starting structure of the substance to be investigated for the HPLC / MS should be known in these experiments. Since the HPLC / MS alone is not suitable for the absolute structure determination. However, if the structure of the parent compound is known, statements can be made about the structure of any metabolites. Since the structure of the substance to be developed as an active ingredient is known, statements about the structure of the unknown metabolites of the drug can be made with some certainty. However, the statement is hampered or prevented by possible overlays other than impurities existing compounds of the same mass. Quantification of the compounds is not possible with this method.

In US 2001/052569 and GB 2 363249 For example, improved methods for parent ion scanning are disclosed.

Furthermore, in US 6,140,638 describes a method of reducing isobaric interference signals by constructing a filter on the collision cell by applying an appropriate field that excludes at least some precursor and intermediate ions that would otherwise result in isobaric interference.

Identification and quantification of a large number or all of the individual components in a substance mixture without available pure substances is still an unsolved problem in mass spectrometry today.

It was therefore the object of a method for analyzing a variety of compounds and preferably to develop their quantification.

1. a) Auswählen mindestens eines Masse/Ladungs-Quotienten (m/z) eines durch Ionisation entstandenen Ionen in einem ersten analytischen Quadrupol (I) des Massenspektrometers,
2. b) Fragmentieren des(r) unter (a) ausgewählten Ions(en) unter Anlegung einer Beschleunigungsspannung in einem weiteren folgenden Quadrupol (II), das mit einem Kollisionsgas gefüllt ist und als Kollisionskammer fungiert,
3. c) Auswählen eines Masse/Ladungs-Quotient eines durch die Fragmentierung (b) entstandenen Fragment-Ions in einem weiteren nachfolgenden Quadrupol (III) und dessen Analyse, wobei die Verfahrensschritte (a) bis (c) mindestens einmal durchlaufen werden und
4. d) Analysieren der Masse/Ladungs-Quotienten aller im Substanzgemisch durch die Ionisation vorhandenen Ionen, wobei das Quadrupol (II) mit Kollisionsgas gefüllt ist, jedoch während der Analyse keine Beschleunigungsspannung angelegt ist;

wobei die Schritt (a) bis (c) und der Schritt (d) auch in umgekehrter Reihenfolge durchgeführt werden können.This object was achieved by a mass spectrometric method for the analysis of substance mixtures with a triple quadrupole mass spectrometer, wherein the substance mixtures before Ionized analysis, characterized in that the method comprises the following steps:

1. a) selecting at least one mass / charge quotient (m / z) of an ion produced by ionization in a first analytical quadrupole (I) of the mass spectrometer,
2. b) fragmenting the ion (s) selected under (a) under application of an acceleration voltage in another following quadrupole (II) which is filled with a collision gas and acts as a collision chamber,
3. c) selecting a mass / charge quotient of a fragment ion formed by the fragmentation (b) in a further subsequent quadrupole (III) and analyzing it, the method steps (a) to (c) being run through at least once, and
4. d) analyzing the mass / charge quotients of all ions present in the mixture of substances by the ionization, the quadrupole (II) being filled with collision gas, but no acceleration voltage being applied during the analysis;

wherein steps (a) to (c) and step (d) may also be performed in reverse order.

Under substance mixtures according to the invention are in principle all mixtures containing more than one substance to understand, such as complex reaction mixtures of chemical syntheses such as synthesis products from combinatorial chemistry or substance mixtures of biological origin such as fermentation broths of an aerobic or anaerobic fermentation, body fluids such as blood, lymph Urine or stool, reaction products a biotechnological synthesis with one or more free or bound enzymes, extracts of animal material such as extracts from various organs or tissues or plant extracts such as extracts of the entire plant or individual organs such as root, stalk, leaf, flower or seed or their mixtures. Advantageously, substance mixtures of biological origin, such as extracts of animal or plant origin, advantageously of plant origin, are analyzed in this process.

The mass spectrometers usable in the method generally comprise a sample inlet system, an ionization chamber, an interface, an ion optics, one or more mass filters and a detector.

In principle, all ion sources known to the person skilled in the art can be used to generate ions in the process. Depending on the ion sources used, these ion sources are coupled via a so-called interface to the following components of the mass spectrometer, for example the ion optics, the mass filter (s) or the detector. The interposition of an interface has the advantage that the analysis can be carried out without delay. Furthermore, nonvolatile and / or volatile, preferably nonvolatile substances can be brought directly into the gas phase by the ion source. As a result, it is also possible to carry out prepurifications of substance mixtures via an advantageous chromatographic separation, which have substance flows of different widths in the analysis, since these substance flows can be processed via the interface. The samples to be analyzed or the substances contained therein can thereby also be enriched. Furthermore, a wide range of solvents can be processed with minimal loss of sample.

1. a) Verdampfung der Substanzgemische und Ionisation der Moleküle bzw. des Substanzgemisches in der Gasphase, beispielsweise wie bei der Elektronenstoss-Ionisation (EI), bei der die Moleküle mit einem Elektronenstrahl in einer Ionisationskammer bei niedrigem Druck (<10^-2 Pa) verdampft werden oder wie bei der chemischen Ionisation (CI) mit einem Reaktandgas bei die Ionen bei einem erhöhtem Druck ca. 100 Pa erzeugt werden. Typische Reaktandgase sind beispielsweise Methan, Isobutan, Ammonium, Argon oder Wasserstoff. Wird die chemische Ionisation bei Atmosphärendruck durchgeführt, so spricht man von der sogenannten "Atmospheric-Pressure Chemical Ionization (APCI).
2. b) Desorption der Substanzgemische von einer Oberfläche beispielsweise wie bei der Plasma Desorption (PD), der Liquid Secondary Ion Mass Spectrometry (LSIMS), dem Fast Atom Bombardment (FAB), der Laser Desorption (LD) oder dem Matrix-Assisted Laser Desorption Ionisation (MALDI). Bei all diesen Methoden werden die Substanzgemische durch einfallende energiereiche Partikel (radioaktiver Zerfall, UV-, IR-Photonen, Ar⁺- oder Cs⁺-Ionen, Laserstrahlen) in einer Kollisionskaskade vibratorisch angeregt und dadurch ionisiert.
3. c) Zerstäubung der Substanzgemische im elektrischen Feld, wie bei der Electrospray-Ionisation (ESI). Bei der Zerstäubung der Substanzgemische im elektrischen Feld werden die Proben bei Atmosphärendruck zerstäubt.

Ionization uses essentially three processes to produce the charged particles (ions):

1. a) evaporation of the substance mixtures and ionization of the molecules or the substance mixture in the gas phase, for example as in the electron impact ionization (EI), in which the molecules are vaporized with an electron beam in an ionization chamber at low pressure (<10 ^-2 Pa) or as in the case of chemical ionization (CI) with a reactant gas in which the ions are produced at an elevated pressure of about 100 Pa. Typical reactant gases are, for example, methane, isobutane, ammonium, argon or hydrogen. If the chemical ionization is carried out at atmospheric pressure, so-called "atmospheric pressure chemical ionization (APCI).
2. b) desorption of mixtures of substances from a surface such as in plasma desorption (PD), liquid secondary ion mass spectrometry (LSIMS), fast atom bombardment (FAB), laser desorption (LD) or matrix-assisted laser desorption ionization (MALDI). In all these methods, the substance mixtures are excited vibrationally by incident high-energy particles (radioactive decay, UV, IR photons, Ar ⁺ or Cs ⁺ ions, laser beams) in a collision cascade and thereby ionized.
3. c) Atomization of the substance mixtures in the electric field, as in electrospray ionization (ESI). In the atomization of the substance mixtures in the electric field, the samples are atomized at atmospheric pressure.

Electrospray ionization is a very gentle method. At ESI, ions are continuously formed. This continuous ion formation has the advantage that it can be easily coupled in conjunction with almost any analyzer type and that it can easily be combined with chromatographic separation such as separation by capillary electrophoresis (CE), liquid chromatography (LC) or high pressure liquid chromatography ( HPLC) because it has a good tolerance for high flow rates up to 2 ml / min eluate. The spraying of the eluent is pneumatically supported by a so-called nebulizing gas, for example nitrogen. For this purpose, the gas is blown under a pressure of up to 4 bar, advantageously up to 2 bar from a capillary, which encloses the inlet capillary of the eluent. Even higher pressures are possible in principle. In the upstream chromatographic separation, so-called normal phase (eg silica gel, alumina, aminodeoxyhexitol, aminodeoxy-d-glucose, triethylenetetramine, polyethylene oxide or aminodicarboxy columns) and / or reversed-phase columns are preferably reversed-phase columns. Columns such as columns with a C ₄ , C ₈ or C ₁₈ stationary phase are preferred. Under standard conditions, the electrospray technique leads to the (quasi-) molecular ion due to the extremely gentle ionization. These are usually adducts with ions already present in the sample solution (eg protons, alkali ions and / or ammonium ions). It is furthermore advantageous that multiply charged ions can also be detected so that ions with a molecular weight of up to one hundred thousand daltons can be detected; molecular weights in a range from 1 to 10,000 daltons, preferably in a range of 50, can advantageously be used in the process according to the invention detect up to 8,000 daltons, more preferably in a range of 100 to 4000 daltons. Other exemplary methods include ion spray ionization, atmospheric pressure ionization (APCI) or thermospray ionization.

In the above-mentioned ionization, the ionization process proceeds under atmospheric pressure and is divided essentially into three phases: First, the solution to be analyzed in a strong electrostatic field by generating a potential difference of 2-10 kV, preferably 2-6 kV between the inlet capillary and a counter electrode is generated, sprayed. An electric field between the inlet capillary tip and the mass spectrometer penetrates the analyte solution and separates the ions in an electric field. Positive ions are attracted to the surface of the liquid in so-called positive mode, negative ions in the opposite direction or vice versa Measurements in the so-called positive mode. The positive ions accumulated on the surface are subsequently drawn further in the direction of the cathode. When using spray capillary (NanoSpray) in which the solution to be examined is not pressed by the application of pressure from the capillary, forms a liquid cone, the so-called Taylor cone, since the surface tension of the liquid counteracts the electric field , If the electric field is strong enough, the cone is stable and continuously emits a liquid stream on its syringe. In pressure-assisted spraying of the solution to be investigated (eg with HPLC), the Taylor cone is not so pronounced.

In each case, an aerosol is formed, which consists of analyte and solvent. In the following stage, the desolvation of the formed drops takes place, which leads to the successive reduction of the droplet size. The evaporation of the solvent is effected by thermal action, e.g. by supplying hot inert gas, achieved. By evaporation in cooperation with the electrostatic forces, the charge density on the surface of the sprayed substance mixture droplets constantly increases. If, finally, the charge density or its charge repulsive forces exceed the surface tension of the droplets (so-called Raleigh limit), then (Coulomb explosion) these droplets explode into smaller droplets. This process "solvent evaporation / Coulomb explosion" is repeated several times until finally the ions go into the gas phase. In order to obtain good measurement results, the gas flow in the interface, the applied heating temperature, the flow rate of the heating gas, the pressure of the nebulizing gas and the capillary voltage must be precisely monitored and controlled.

With the different ionization methods, single or multiple charged ions can be generated. For the method according to the invention, the ionization method used is advantageously methods for atomizing the substance mixture in the electric field, such as the thermospray, the electrospray (= ES) or the atmospheric pressure chemical ionization (= APCI) method. In APCI ionization, ionization occurs in a so-called corona discharge. Preference is given to the thermospray or electrospray method, the electrospray method being particularly preferred. The ionization space is connected via an interface, that is to say via a micro-opening (100 μm) with the following mass spectrometer. On the side of the ionization chamber, an interface plate with a larger opening is still attached. Between this plate and the so-called "orifice" is a heated Carrier gas (= Curtain gas), for example, injected nitrogen. The nitrogen collides with the ions generated, for example, by electrospray, which were generated in the substance mixture. By blowing the curtain gas is advantageously prevented that neutral particles are sucked into the high vacuum of the subsequent mass spectrometer. Furthermore, the curtain gas promotes the desolvation of the ions.

The method according to the invention can be carried out with all quadrupole mass spectrometers known to the person skilled in the art, such as the triple quadrupole mass spectrometers. In US 2,939,952 describes and claims Paul et al. a first such device. These devices have a favorable mass range up to about m / z = 4000 and achieve resolution values between 500 and about 5000. They have high ion transmission from the source to the detector, are easy to focus and calibrate, and advantageously have great stability Calibration in continuous operation. Triple quadrupole instruments are the standard tools for low energy collision activation studies. Usually, these devices consist of a first quadrupole, which is suitable for analyzing the mass / charge quotient (m / z) of the ions contained in the substance mixture after ionization in a high vacuum (about 10 ^-5 Torr), the mass (s) individual ions, several or all ions can be measured. This first analytical quadrupole (= I or Q1) can be preceded by one or more quadrupoles (= Q0), which are usually used to focus the ions. Instead of this or these upstream quadrupoles, it is also possible to use so-called "cones" lenses or lens systems for focusing and introducing the ions into the first analytical quadrupole. Also combinations of quadrupoles and cones are realized and usable.

Another Q1 following quadrupole (= II or Q2) serves as a collision chamber. In it, the ions are advantageously fragmented by applying a fragmentation voltage. For fragmentation, ionization potentials in the range of 5-11 electron volts (eV), preferably 8-11 electron volts (eV) are applied. Q2 is also filled for fragmentation in the process according to the invention with a collision gas such as a noble gas such as argon or helium or other gas such as CO ₂ or nitrogen or mixtures of these gases such as argon / helium or argon / nitrogen. For cost reasons, argon and / or nitrogen is preferred. In the collision chamber, the collision gas is present in the process according to the invention at a pressure of 1 × 10 ^-5 to 1 × 10 ^-1 Torr, preferably 10 ^-2 . Particularly preferred is nitrogen. Even without the application of a fragmentation voltage, there may be a scattered fragmentation of the ions in the collision chamber in the presence of a Collision gases come. Between quadrupole Q1 and Q2 there may be additional quadrupoles or cones for guiding the ions.

The quadrupole Q2, which serves as a collision chamber, is followed by another quadrupole (= III or Q3). In this Q3, either the m / z quotients of individual selected fragments, several or all of the m / z quotients present in the substance mixtures after ionization (in this application for simplicity's sake referred to as mass or masses) can be determined. Also between quadrupole Q2 and Q3 there may be more quadrupoles or cones for steering the ions.

In the method according to the invention, individual quadrupoles for collecting ions can also be operated as ion traps, from which the ions are then released again for analysis after some time.

The quadrupoles used in the triple quadrupole mass spectrometers generate a three-dimensional electric field in which the generated ions can be held. They usually consist of 4, 6 or 8 rods or rods with their help an oscillating electric field is generated, opposite rods are electrically connected. In addition to the name quadrupole, the terms hexa- or octapol are also used. In the present application, these terms should be included when the term quadrupole is used. Advantageously, only low acceleration voltages of a few volts, preferably of a few 10 volts, are required in the quadrupoles of the triple quadrupole mass spectrometer for guiding the ions.

Substance mixtures such as animal or vegetable extracts, preferably vegetable extracts, are advantageously used in the process according to the invention.

1. I) In den Verfahrensschritten (a) bis (c) wird die Masse mindestens eines im Substanzgemisch nach Ionisation in Q1 vorhandenen Ions analysiert und ausgewählt. Dieses ausgewählte Ion wird anschließend in Q2 in Gegenwart von Kollisionsgas und einer Fragmentierungsspannung fragmentiert und danach wird eines der entstandenen Fragment-Ionen in einem weiteren analytischen Quadrupol Q3 identifiziert und vorteilhaft auch quantifiziert. Dabei erfolgt die Auswahl des zu analysierenden Fragment-Ions in der Weise, dass diese Ion vorteilhaft eine hohe Intensität, eine leicht identifizierbare charakteristische Masse hat und in einer vorteilhaften Ausführungsform des Verfahrens eine leichte Quantifizierung ermöglicht.
2. II) Anschließend werden im Verfahrensschritt (d) die Massen aller im Substanzgemisch nach Ionisation vorhandenen Ionen analysiert, wobei das als Kollisionskammer benutzte Quadrupol Q2 immer mit Kollisionsgas gefüllt ist, jedoch in Verfahrensschritt (d) keine Fragmentierungsspannung an Q2 anliegt. Diese Analyse kann prinzipiell sowohl mit Q1 und als auch mit Q3 erfolgen, vorteilhafter ist es jedoch die Analyse mit Q3 durchzuführen, da zwischen Q1 und dem an das Massenspektrometer anschließenden Detektor als Kollisionskammer verwendete Quadrupol Q2 liegt. Sollte in Q2 eine Fragmentierung trotz dem fehlen anliegender Fragmentierungsspannung auftreten, so hat dies keinen Einfluss auf eine mögliche Erfassung der Ionenmassen am Detektor. Im Falle einer Massenanalyse mit Q1 würde eine solche Fragmentation in Q2 jedoch zu Fehlschlüssen bei der Detektion führen. Deshalb ist eine Massendetektion mit Q3 bevorzugt, da mögliche Fehlerquellen eliminiert werden bzw. vernachlässigbar sind.

In the process according to the invention, the further process steps are carried out after the ionization of the substance mixtures

1. I) In method steps (a) to (c), the mass of at least one ion present in the substance mixture after ionization in Q1 is analyzed and selected. This selected ion is then fragmented in Q2 in the presence of collision gas and a fragmentation voltage and then one of the resulting fragment ions in another analytical quadrupole Q3 is identified and advantageously also quantified. In this case, the selection of the fragment ion to be analyzed in such a way that this ion is advantageously a high intensity, a has easily identifiable characteristic mass and in an advantageous embodiment of the method enables easy quantification.
2. II) Subsequently, in method step (d), the masses of all ions present in the substance mixture after ionization are analyzed, wherein the quadrupole Q2 used as a collision chamber is always filled with collision gas, but in method step (d) no fragmentation voltage is applied to Q2. This analysis can in principle be carried out with both Q1 and Q3, but it is more advantageous to carry out the analysis with Q3, since quadrupole Q2 is used as the collision chamber between Q1 and the detector following the mass spectrometer. Should fragmentation occur in Q2 despite the lack of fragmentation voltage, this does not affect the possible detection of ion masses at the detector. However, in the case of mass analysis with Q1, such fragmentation in Q2 would lead to erroneous detection. Therefore, mass detection with Q3 is preferred since possible sources of error are eliminated or negligible.

The process steps (I) and (II) listed above can also be carried out in the reverse order. FIG. 1 the sequence of the method according to the invention can be seen. In the process according to the invention, process steps (a) to (c) and (d) are advantageously carried out at least once within 0.1 to 10 seconds, preferably within 0.2 to 6 seconds at least once, particularly preferably within 0.2 to 2 Seconds, most preferably at least once within 0.3 to less than 2 seconds. In order to enable an advantageous statistical evaluation of the measurements, the process steps are run through within two to three times, preferably three times, within 0.2 to 6 seconds. In order to enable such rapid measurements following one after the other, the quadrupole Q2 acting as a collision chamber is constantly filled with collision gas. As the own measurements showed, this has no negative influence on the reproducibility of the measurements.

During an analysis, between 1 and 100 mass / charge quotients of different ions formed and selected in step (a) can be analyzed in the method according to the invention. Advantageously, at least 20 m / z quotients, preferably at least 40 m / z quotients, more preferably at least 60 m / z quotients, most preferably at least 80 m / z quotients of different ions or more are identified and / or quantified.

With the aid of the method according to the invention, in addition to the analysis of all masses present in a substance mixture, it is also possible advantageously to analyze and advantageously quantify individual substances or their masses.

Purification of the substance mixtures is in principle not required in the process according to the invention. The substance mixtures can be measured directly after introduction into an ion source. This also applies to complex mixtures of substances. It is also not necessary to add to the substance mixtures as internal standards any labeled or unlabeled pure substances of possible substances contained in the mixtures, although this is of course possible and simplifies subsequent quantification of the substances contained in the mixtures.

However, purification by methods known to a person skilled in the art, such as chromatographic methods, is advantageous. Because of the preferred in the process according to the invention ionization method on a sputtering of the substance mixtures in the electric field, an up and / or pre-purification of the substance mixtures can be coupled very easily, for example via chromatography to the mass spectrometric analysis. In this case, all separation methods known to the person skilled in the art, such as LC, HPLC or capillary electrophoresis, can be used as chromatographic methods. Separation methods based on adsorption, gel permeation, ion pair, ion exchange, exclusion, affinity, normal phase or reversed phase chromatography, to name a few possible ones, can be used. Advantageously, normal-phase and / or reversed-phase-based chromatographies, preferably reversed-phase columns with different hydrophobic modified materials such as C ₄ -, C ₈ -, or C ₁₈ - phases are used.

In the process according to the invention, coupling of purification methods, advantageously by chromatography methods with a flow rate of the eluent (analyte + solvent), is advantageously between 1 μl / min to 2000 μl / min, preferably between 5 μl / min to 600 μl / min, more preferably between 10 μl / min to 500 .mu.l / min, for example, possible. Even lower or higher flow rates can be used without difficulty in the process according to the invention.

In principle, all protic or aprotic polar or non-polar solvents which are compatible with the subsequent analysis can be used as solvents for the purification process. Whether a solvent is compatible with mass spectrometry, the skilled person can easily determine by simple random tests. Suitable solvents are, for example Solvents carrying little or no charge, such as aprotic apolar solvents, characterized by a low dielectric constant (E _t <15), low dipole moments (μ <2.5D), and low E _T ^N values (0.0-0.5 ) are characterized. But also dipolar organic solvents or mixtures thereof are suitable as solvents for the inventive method. Examples of suitable solvents are methanol, ethanol, acetonitrile, ethers, heptane. Also weak acid solvents such as 0.01 - 0.1% formic acid, acetic acid or trifluoroacetic acid are suitable. Furthermore, weakly basic solvents such as 0.01 to 0.1% triethylamine or ammonia are also suitable. Strongly acidic or strongly basic solvents such as 5% HCl or 5% triethylamine are also suitable in principle as solvents. Also, mixtures of the aforementioned solvents are advantageous. The customary in biochemical buffers are suitable as solvents, wherein advantageously buffer <200 mM, preferably <100 mM, more preferably <50 mM, most preferably <20 mM are used. It is also advantageous if buffers> 100 mM are used for the preparation of the substance mixtures that the buffers are completely or partially removed, for example via dialysis. Examples of suitable buffers include acetate, formate, phosphate, tris, MOPS, HEPES or mixtures thereof. High buffers and / or salt concentrations have a negative effect on the ionization processes and may need to be avoided.

In the process according to the invention, molecules contained in the substance mixtures can be from 100 daltons (= D) to 100 kilodaltons (= kD), preferably from 100 D to 20 kD, more preferably from 100 D-10 kD, most preferably from 100 D to prove 2000 D, ie identify and possibly also quantify.

Advantageously, the substance mixtures for the method according to the invention, which are otherwise poorly or not at all detectable, can be derivatized before the analysis and thus finally analyzed. Derivatization is particularly advantageous in cases in which hydrophilic groups are introduced into hydrophobic or volatile compounds, for example, such as esters, amides, lactones, aldehydes, ketones, alcohols, etc., which advantageously still carry an ionizable functionality. Examples of such derivatizations are reactions of aldehydes or ketones to oximes, hydrazones or their derivatives or alcohols to give esters, for example with symmetrical or mixed anhydrides. As a result, the detection spectrum of the method can advantageously be extended.

Advantageously, in the method according to the invention for analyzing the substance mixtures, an internal standard, such as e.g. Peptides, amino acids, coenzymes, sugars, alcohols, conjugated alkenes, organic acids or bases added. This internal standard advantageously allows the quantification of the compounds in the mixture. Substance mixture containing substances can thus be more easily analyzed and ultimately quantified.

As an internal standard, advantageously labeled substances are used, but in principle also non-labeled substances are suitable as an internal standard. Such similar chemical compounds are, for example, so-called compounds of a homologous series whose members differ only by, for example, an additional methylene group. As an internal standard, at least one isotope selected from the group ² is preferably H, ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³³ S, ³⁴ S, ³⁶ S, ³⁵ Cl, ³⁷ Cl, ²⁹ Si, ³⁰ Si, ⁷⁴ Se or mixtures thereof used labeled substances. For cost reasons and for reasons of accessibility, ² H or ¹³ C is preferably used as the isotope. This internal standard does not need to be complete for the analysis, that is, to be fully marked. A partial marking is completely sufficient. In the case of a labeled internal standard, it is also advantageous to choose a substance which has the highest possible homologs to the substances to be analyzed in the mixture, that is to say structural similarity, to the chemical compound to be measured. The higher the structural similarity, the better the measurement results and the more accurate the quantification of the compound.

For the process according to the invention and especially for the quantification of the substances present in the mixture, it is advantageous to use the internal standard in a favorable ratio to the substance to be measured. Ratios of analyte (= compound to be determined) to internal standard greater than 1:15 lead to no improvement in the measurement results, but are possible in principle. Advantageously, a ratio of analyte to internal standard is set in a range from 10: 1 to 6: 1, preferably in a range from 6: 1 to 4: 1, particularly preferably in a range from 2: 1 to 1: 1.

The substance mixture samples in the process according to the invention can be prepared manually or advantageously automatically with conventional laboratory robots. The analysis with the mass spectrometer after optional chromatographic separation can be carried out manually or advantageously automatically. By automating the process according to the invention, mass spectrometry can advantageously be used for the rapid screening of various substance mixtures, for example plant extracts in the so-called high-throughput screening. In this case, the method according to the invention is characterized by a high sensitivity, a good quantifiability, an excellent reproducibility, with the lowest sample consumption. The method can thus rapidly mixtures of biological origin, for example, new mutants known or unknown enzymatic activities after a mutagenesis, for example, a classical mutagenesis with chemical agents such as NTG, radiation such as UV radiation or X-rays or after a so-called site-directed mutagenesis, PCR mutagenesis , Transposon mutagenesis or so-called gene shuffling.

The method according to the invention makes it possible to analyze a broad range of substances in a wide measuring range, with good to very good resolution, with a high ion transmission from the source to the detector, a high scanning speed both in the full scan mode of all substances in the substance mixtures [ = FS, process step (d)) as well as in the multiple reaction monitoring mode [= MRM, process steps (a) to (c)]. Furthermore, the method has a very high recording sensitivity and excellent calibration stability. Furthermore, it is excellently suitable for continuous operation and thus for use in HTS screening.

The invention is further illustrated by the following examples:

Examples 1. Examples: MRM + FS measurements a) TIC of the MRM + FS measurement

In FIG. 2 is the Total Ion Chromatogram of an MRM + Full Scan Measurement [MRM - Multiple Reaction Monitoring, FS - Full Scan, TIC - Total Ion Chromatogram, XIT = Sum of Multiple Total Ion Chromatograms]. A quality control sample was measured. This type of sample contains a defined number of analytes. These analytes were purchased and dissolved in known concentrations in appropriate solvent.

In the FIG. 2 The selected representation of the measurement shows the summation of the intensities (y-axis) measured at the detector at the respective times (x-axis) from the two mass-spectrometric experiments of Multiple Reaction Monitoring (MRM) and Full Scan (FS). The chromatogram in FIG. 2 thus represents the sum of the TIC chromatograms of the above-mentioned mass spectrometric experiments.

b) TIC of the MRM experiment

In FIG. 3 the total ion chromatogram of the MRM experiment from an MRM + FS measurement is shown.

In the FIG. 3 The selected representation of the MRM measurement shows the summation of the intensities (y-axis) measured at the detector at the respective times (x-axis) from all predefined mass transitions of the MRM experiment. In the FIG. 4 selected representation shows the respective measurement results of each mass transition (here 30 pieces) in a coordinate system.

c) TIC of the FS experiment

The FS experiment measured in change to the MRM experiment is in the TIC in FIG. 5 shown.

In FIG. 6 the TIC of the FS experiment is shown. The summation of all FS mass spectra recorded in the time window shown hatched are in FIG. 7 shown.

d) TIC of an MRM experiment

In FIG. 8 is like in FIG. 3 a total ion chromatogram of an MRM + full scan measurement. A calibration sample was measured.

In the FIG. 8 The selected representation of the measurement shows the summation of the intensities (y-axis) measured at the detector at the respective times (x-axis) from the mass-spectrometric experiment of multiple reaction monitoring.

FIG. 9 returns an extracted chromatogram identifying coenzyme Q 10.

FIG. 10 and FIG. 11 give the identification of each capsanthin and bixin again.

FIG. 12 returns a Total Ion Chromatogram of a full scan of a plant extract.

The FIGS. 13 to 15 show the masses of different analytes in the extracted chromatogram whose assignment to a specific structure has yet to be made.

So far, 200 additional analytes have been selectively detected in the described method.

Figur 1:

Schematische Darstellung des Analysenverfahrens Nachdem in Prozessschritt I die Verfahrensschritte (a) bis (c) durchlaufen werden wird in Prozessschritt II der Verfahrensschritt (d) durchlaufen, und umgekehrt

Figur 2:

Darstellung der Summe der Total Ion Chromatogramme (TIC) einer Multiple Reaction Monitoring (MRM)-Messung (Verfahrensschritte (a) bis (c) des Verfahrens) und Full Scan(FS)-Messung (Verfahrensschritt (d) des Verfahrens) von Probe 2

Figur 3:

Darstellung des Total Ion Chromatogramms (TIC) bzgl. des Multiple-Reaction-Monitoring(MRM)-Parts (Verfahrensschritte (a) bis (c) des Verfahrens) mit 30 vordefinierten Massenübergängen (sogenannte Pairs), wobei die Messung selbst sowohl auf einer MRM-Messung (Verfahrensschritte (a) bis(c)) als auch FS-Messung (Verfahrensschritt (d)) von Probe 2 beruht

Figur 4:

Darstellung des Total Ion Chromatogramms (TIC) bzgl. des Multiple-Reaction-Monitoring(MRM)-Parts (Verfahrensschritte (a) bis (c) des Verfahrens) mit 30 vordefinierten Massenübergängen (sogenannte Pairs), wobei die Messung selbst sowohl auf einer MRM-Messung (Verfahrensschritte (a) bis(c)) als auch FS-Messung (Verfahrensschritt (d)) von Probe 2 beruht, und wobei jedes Pair einzeln dargestellt ist

Figur 5:

Darstellung des Total Ion Chromatogramms (TIC) bzgl. des FS-Parts (Verfahrensschritt (d) des Verfahrens), wobei die Messung selbst sowohl auf einer MRM-Messung (Verfahrensschritte (a) bis(c)) als auch FS-Messung (Verfahrensschritt (d)) von Probe 2 beruht

Figur 6:

Darstellung des Total Ion Chromatogramms (TIC) bzgl. des FS-Parts (Verfahrensschritt (d) des Verfahrens), wobei die Messung selbst sowohl auf einer MRM-Messung (Verfahrensschritte (a) bis(c)) als auch FS-Messung (Verfahrensschritt (d)) von Probe 2 beruht, wie in Figur 5, wobei ein Zeitfenster schraffiert hervorgehoben ist

Figur 7:

Darstellung des Total Ion Chromatogramms (TIC) bzgl. des FS-Parts (Verfahrensschritt (d) des Verfahrens), welches in dem in Figur 6 schraffiert dargestellten Zeitfenster (1,491 bis 2,004 min) aufgenommen wurde, wobei die Messung selbst sowohl auf einer MRM-Messung (Verfahrensschritte (a) bis(c)) als auch FS-Messung (Verfahrensschritt (d)) von Probe 2 beruht

Figur 8:

Darstellung des Total Ion Chromatogramms (TIC) bzgl. des Multiple-Reaction-Monitoring(MRM)-Parts (Verfahrensschritte (a) bis (c) des Verfahrens) mit 36 vordefinierten Massenübergängen (sogenannte Pairs), wobei die Messung selbst sowohl auf einer MRM-Messung (Verfahrensschritte (a) bis(c)) als auch FS-Messung (Verfahrensschritt (d)) einer Kalibrierungsprobe beruht

Figur 9:

Darstellung des für den Massenübergang m/z 863.7 nach 197 (Coenzym Q10) extrahierten Total Ion Chromatogramms einer Kalibrierungsprobe wobei der MRM-Part 36 Pairs umfaßte

Figur 10:

Darstellung des für den Massenübergang m/z 585.4 nach 109.1 (Capsanthin) extrahierten Total Ion Chromatogramms einer Kalibrierungsprobe, wobei der MRM-Part 36 Pairs umfaßte

Figur 11:

Darstellung des für den Massenübergang m/z 395.1 nach 91.1 (Bixin) extrahierten Total Ion Chromatogramms einer Kalibrierungsprobe, wobei der MRM-Part 36 Pairs umfaßte

Figur 12:

Darstellung des Total Ion Chromatogramm (TIC) eines Pflanzenextrakts bzgl. des FS -Parts (Verfahrensschritt (d) des Verfahrens)

Figur 13:

Darstellung des für m/z = 518.4 extrahierten Total Ion Chromatogramms von Probe 4

Figur 14:

Darstellung des für m/z = 609.2 extrahierten Total Ion Chromatogramms von Probe 4

Figur 15:

Darstellung des für m/z = 210.0 extrahierten Total Ion Chromatogramms von Probe 4

Characters:

FIG. 1:

Schematic representation of the analytical process After process steps (a) to (c) have been carried out in process step I, process step (d) is run through in process step II, and vice versa

FIG. 2:

Representation of the sum of Total Ion Chromatograms (TIC) of a Multiple Reaction Monitoring (MRM) measurement (process steps (a) to (c) of the process) and Full Scan (FS) measurement (process step (d) of the process) of Sample 2

FIG. 3:

Representation of the total ion chromatogram (TIC) with respect to the multiple reaction monitoring (MRM) part (method steps (a) to (c) of the method) with 30 predefined mass transitions (so-called pairs), the measurement itself being carried out on an MRM Measurement (method steps (a) to (c)) as well as FS measurement (method step (d)) of sample 2

FIG. 4:

Representation of the total ion chromatogram (TIC) with respect to the multiple reaction monitoring (MRM) part (method steps (a) to (c) of the method) with 30 predefined mass transitions (so-called pairs), the measurement itself being carried out on an MRM Measurement (method steps (a) to (c)) and FS measurement (method step (d)) of sample 2, and wherein each pair is shown individually

FIG. 5:

Representation of the total ion chromatogram (TIC) with respect to the FS part (method step (d) of the method), the measurement itself being measured both on an MRM measurement (method steps (a) to (c)) and FS measurement (method step (d)) of sample 2

FIG. 6:

Representation of the total ion chromatogram (TIC) with respect to the FS part (method step (d) of the method), the measurement itself being measured both on an MRM measurement (method steps (a) to (c)) and FS measurement (method step (d)) is based on sample 2, as in FIG. 5 , where a time window is highlighted hatched

FIG. 7:

Representation of the total ion chromatogram (TIC) with respect to the FS part (method step (d) of the method), which is described in the in FIG. 6 The measurement itself is based on both an MRM measurement (method steps (a) to (c)) and FS measurement (method step (d)) of sample 2

FIG. 8:

Representation of the Total Ion Chromatogram (TIC) with respect to the Multiple Reaction Monitoring (MRM) Part (method steps (a) to (c) of the method) with 36 predefined mass transitions (so-called pairs), the measurement itself being carried out on an MRM Measurement (method steps (a) to (c)) as well as FS measurement (method step (d)) of a calibration sample

FIG. 9:

Representation of the Total Ion Chromatogram of a Calibration Sample Extracted for Mass Transfer m / z 863.7 to 197 (Coenzyme Q10) where the MRM Part comprised 36 pairs

FIG. 10:

Representation of the total ion chromatogram of a calibration sample extracted for mass transfer m / z 585.4 to 109.1 (capsanthin), the MRM part comprising 36 pairs

FIG. 11:

Representation of the total ion chromatogram of a calibration sample extracted for mass transfer m / z 395.1 to 91.1 (bixin), with the MRM part comprising 36 pairs

FIG. 12:

Representation of the total ion chromatogram (TIC) of a plant extract with regard to the FS part (method step (d) of the method)

FIG. 13:

Representation of the total ion chromatogram of sample 4 extracted for m / z = 518.4

FIG. 14:

Representation of the total ion chromatogram extracted from sample 4 for m / z = 609.2

FIG. 15:

Representation of the total ion chromatogram of sample 4 extracted for m / z = 210.0

Claims (13)

1. A mass spectrometry process for analyzing substance mixtures using a triple quuadrupole massspectrometer, said substance mixtures being ionized before the analysis, which comprises the following steps:

   a) selecting at least one mass/charge quotient (m/z) of ions formed by ionization in a first analytical quadrupole (I) of the mass spectrometer,

   b) fragmenting the ion(s) selected under (a) by applying an acceleration voltage in a further following quadrupole (II) which is filled with a collision gas and functions as a collision chamber,

   c) selecting and analyzing a mass/charge quotient of a fragment ion formed by the fragmentation (b) in a further downstream quadrupole (III), the process steps (a) to (c) being run through at least once, and

   d) analyzing the mass/charge quotients of all ions present in the substance mixture as a result of the ionization, the quadrupole (II) being filled with collision gas but no acceleration voltage being applied during the analysis;

   and the steps (a) to (c) and step (d) may also be carried out in reverse sequence.
2. The process according to claim 1, wherein the ionization of the substance mixture is downstream of a chromatographic separation.
3. The process according to claim 1 or 2, wherein the chromatographic separation is a high pressure chromatography (HPLC) separation.
4. The process according to claims 1 to 3, wherein steps (a) to (d) are run through at least once within from 0.1 to 10 seconds.
5. The process according to claims 1 to 4, wherein steps (a) to (d) are run through at least once within from 0.2 to 2 seconds.
6. The process according to claims 1 to 5, wherein the ionization is effected by evaporating the substance mixture and ionizing in the gas phase, by desorbing the substance mixture on a surface or by atomizing the substance mixture in an electrical field.
7. The process according to claims 1 to 6, wherein the ionization is effected by atomizing the substance mixture in an electrical field.
8. The process according to claims 1 to 7, wherein in step (a) between 1 and 100 mass/charge quotients of different ions formed by ionization and selected, are analysed.
9. The process according to claims 1 to 8, wherein the substance mixture is of biological or chemical origin.
10. The process according to claims 1 to 9, wherein the substance mixtures are derivatized before the analysis or before the chromatographic separation according to claim 2 or 3.
11. The process according to claims 1 to 10, which is carried out manually or automatically.
12. The process according to claims 1 to 11, which is used in a high-throughput screening.
13. The process according to claims 1 to 12, wherein

    (i) the fragment ion analyzed in step (c) and the (m/z) quotients, analyzed in step (d), of all ions present in the substance mixture, or

    (ii) the fragment ion analyzed in step (c), or

    (iii) the (m/z) quotients, analyzed in step (d), of all ions present in the substance mixture
    are quantified.

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