EP1481416A1

EP1481416A1 - Mass spectrometry method for analysing mixtures of substances

Info

Publication number: EP1481416A1
Application number: EP03711878A
Authority: EP
Inventors: Tilmann B. Walk; Martin Dostler
Original assignee: Metanomics GmbH
Current assignee: BASF Metabolome Solutions GmbH
Priority date: 2002-02-28
Filing date: 2003-02-10
Publication date: 2004-12-01
Anticipated expiration: 2023-02-10
Also published as: CA2476597A1; US7196323B2; AU2003218649B2; NO20043943L; JP2014041142A; WO2003073464A1; EP1481416B1; CA2476597C; JP2010019848A; ES2590759T3; IL163290A; US20050103991A1; AU2003218649A1; JP2005526962A

Abstract

The invention relates to a mass spectrometry method for analysing mixtures of substances using a triple quadrupole mass spectrometer, whereby said mixtures of substances are ionised prior to analysis. The invention is characterised in that the method comprises the following steps: a) selection of a mass/charge quotient (m/z) of an ion created by ionisation in a first analytical quadrupole (I) of the mass spectrometer; b) fragmentation of the ion selected in step (a) by applying an acceleration voltage in an additional subsequent quadrupole (II), which is filled with a collision gas and acts as a collision chamber; c) selection of a mass/charge quotient of an ion created by the fragmentation process in step (b) in an additional subsequent quadrupole (III), whereby steps (a) to (c) of the method are carried out at least once; and d) analysis of the mass/charge quotients of all the ions present in the mixture of substances as a result of the ionisation process, whereby the quadrupole (II) is filled with collision gas, but no acceleration voltage is applied during the analysis. Steps (a) to (c) and step (d) can also be carried out in reverse order.

Description

Mass spectrometric method for the analysis of substance mixtures

description

The present invention relates to a mass spectrometric method for analyzing substance mixtures using a triple quadrupole mass spectrometer.

When analyzing complex substance mixtures of biological and / or chemical origin, in addition to the task of identifying the structure of individual substances contained in the mixture, the analyst repeatedly faces the problem of detecting and quantifying as far as possible all substances present in the mixture. This should be done as quickly as possible and with a high degree of accuracy, i.e. with a small error deviation. This becomes all the more important when information about a biological system is to be obtained, for example, about a microorganism grown under certain fermentation conditions or about a plant grown under different environmental conditions or about a wild-type organism such as a microorganism or a plant in comparison with its genetically modified mutant , Such comparisons are necessary in order to enable mutations of unknown genes in the genome of these organisms to be assigned to a specific metabolic phenotype.

The success in the analysis of this substance mixture, for example chemical synthesis approaches from combinatorial chemistry or from extracts of microorganisms, plants or parts of plants, depends to a large extent on the speed and reproducibility of the analytics used. In such a screening, a large number of samples have to be screened. Fast, simple, highly sensitive and highly specific analysis methods are therefore required.

A major problem with this analysis is the quick, simple, reproducible and quantifiable identification of the substances contained in the mixtures. Separation methods such as thin layer chromatography (= DC), high pressure liquid chromatography (= HPLC) or gas chromatography (= GC) are generally used to analyze the products. However, a wide range of substances cannot be identified and quantified quickly and easily using these chromatographic methods. Methods such as NMR or mass spectrometry are also described for this task. As a rule, however, there is some preparation of the samples for these analysis procedures 2 required, such as processing via salt precipitation and / or subsequent chromatography, concentration, desalination 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 are not used in so-called high-throughput screening (= HTS) or the broad screening of substance mixtures in biological or chemical samples. The advantage of very precise methods such as NMR or IR spectroscopy is that they provide information about both the structure and, if appropriate, the quantity of a substance.

In order to enable a higher sample throughput in the HTS, indirect, easily measurable methods such as color reactions in the visible range, turbidity measurements, fluorescence, conductivity measurements etc. are often used. In principle, these are very sensitive, but they are also prone to failure. The main disadvantage here is that many false positive samples are analyzed with this procedure and, since these 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 procedure, further analysis methods are usually used after a first rapid analysis, such as NMR, IR, HPLC / MS or GC / MS. Again, this is very time consuming.

In general, it can be said that the improvement in the sensitivity and the informative value of the detection methods leads to a slowdown in the speed of an analysis.

When working with complex biological mixtures, such as extracts from microorganisms, plants and / or animals, it should also be noted that individual compounds are only present in the mixtures in very small quantities or only small quantities of the individual sample itself for analysis are available so that the method used must be highly sensitive. Furthermore, the non-volatile buffers and / or salts frequently present in biological samples pose a problem for some analytical methods, since these negatively influence the sensitivity of the methods or their use at all. 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, which range, for example, from the analysis of samples from synthetic chemistry, petrochemistry, environmental samples and biological material. However, these methods are only used for the analysis of individual known compounds in these samples. Wide series of measurements, for example in the context of an HTS or in the identification and quantification of a large number of compounds in these samples, are not described.

The coupling of gas chromatography and mass spectrometry (= GC / MS) is used for substances that are extractable from the substance mixtures and are volatile. So-called liquid chromatography or high-pressure liquid chromatography is used for the analysis of substances or analytes that cannot be converted into the gas phase easily or only with difficulty and for which a large excess of solvent has to be removed -Mass spectrometry (= HPLC / MS) used. An overview of the various LC / MS methods and their equipment can be found in the publication by Niessen et al. (Journal of Chromatography A, 703, 1995: 37-57). Mass spectrometers and their structure are described and claimed in US Pat. Nos. 4,540,884 and 5,397,894.

With the help of the aforementioned methods, substances in a molecular weight range of up to 100 KD (= 'KiloDalton) can be determined, i.e. a wide range of substances, for example in a lower mass range of up to about 5000 D (= Dalton), such as fatty acids, Determine amino acids, carboxylic acids, oligo- 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 kerogens can also be analyzed (Zenobie and Knochenmuss, 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 that it has to be confirmed using other methods, for example NMR.

G. Hopfgartner and F. Vilbois (Analusis, 2001, 28, No. 10, 906-914) describe a method for screening with the aid of LC / MS of metabolites of structurally known compounds which are formed in vitro or in vivo and which are known as Active ingredients are in different phases of drug development. This process takes place in two steps. In the first search step, interesting ions are detected in a rapid "fill scan mode", which can be considered as candidates for further investigations. men. These can be ions which correspond to ions of particularly high intensity or can be considered as candidates for possible degradation products or metabolites of the active ingredients. 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. The collision chamber constantly contains collision gas in order to enable a rapid elucidation of the ion or metabolite structure. A disadvantage of the structure determination is that a known mass of a precursor ion, a fragment or an ion adduct is required. The starting structure of the substance to be investigated for HPLC / MS should advantageously be known in these experiments. Since 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 active ingredient can be made with some certainty. However, the statement is made more difficult or prevented by possible superimposition of compounds of the same mass other than impurities. A quantification of the compounds is not possible with this method.

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

There was therefore the task of a method for analyzing a

Develop a variety of compounds and preferably their quantification.

This object was achieved by a mass spectrometric method for the analysis of substance mixtures with a triple quadrupole mass spectrometer, the substance mixtures being ionized before the analysis, characterized in that the method comprises the following steps:

a) selecting a mass / charge quotient (m / z) of an ion formed by ionization in a first analytical quadrupole (I) of the mass spectrometer, b) fragmenting the ion selected under (a) by applying an acceleration voltage in a further subsequent quadrupole (II) which is filled with a collision gas and acts as a collision chamber,

c) selecting a mass / charge quotient of an ion formed by the fragmentation (b) in a further subsequent quadrupole (III), the process steps (a) to (c) being carried out at least once and

d) analyzing the mass / charge quotients of all the 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) can also be carried out in the reverse order.

Substance mixtures in the sense of the invention are in principle all mixtures which contain more than one substance, for example complex reaction mixtures of chemical syntheses such as synthesis products from combinatorial chemistry or mixtures of substances 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 roots, stems, leaves, flowers or seeds or their mixtures. Mixtures of substances of biological origin, such as extracts of animal or vegetable origin, advantageously of vegetable origin, are advantageously analyzed in this method.

The mass spectrometers that can be used in the method usually consist of a sample inlet system, an ionization chamber, an interface, 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 source 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. Interposing an interface has the advantage that the analysis can be carried out without delay. Furthermore, the ion source cannot volatile and / or volatile, preferably non-volatile substances are brought directly into the gas phase. In this way, pre-purifications of substance mixtures can also be carried out via an advantageous chromatographic separation, which have material flows of different widths in the analysis, since these material flows can be processed via the interface. The samples to be analyzed or the substances contained therein can also be enriched as a result. Furthermore, a wide range of solvents can be processed with the least loss of sample.

Basically, three processes are used to generate the charged particles (ions):

a) Evaporation of the substance mixtures and ionization of the molecules or the substance mixture in the gas phase, for example as in electron impact ionization (EI), in which the molecules evaporate with an electron beam in an ionization chamber at low pressure (<10 ^{~ 2} P _a ) be or as in chemical ionization (CI) with a reactant gas in which the ions are generated at an increased pressure of about 100 Pa. Typical reactant gases are, for example, methane, isobutane, ammonium, argon or hydrogen. If chemical ionization is carried out at atmospheric pressure, one speaks of the so-called "Atmospheric-Pressure Chemical Ionization (APCI).

b) Desorption of the substance mixtures from a surface, for example as in plasma desorption (PD), liquid secondary ion mass spectrometry (LSIMS), fast atom bombardment (FAB), laser desorption (LD) or the matrix-assisted laser Desorption ionization (MALDI). In all of these methods, the substance mixtures are vibrationally excited by incident energy-rich particles (radioactive decay, UV, IR photons, Ar ⁺ or Cs ⁺ ions, laser beams) in a collision cascade and thereby ionized.

c) atomization of the substance mixtures in an electrical field, as in electrospray ionization (ESI). When the substance mixtures are atomized in the electric field, the samples are atomized at atmospheric pressure.

Electrospray ionization is a very gentle method. Ions are continuously formed at ESI. This continuous ion formation has the advantage that it can be easily connected to almost any type of analyzer and that it can be easily combined with a chromatographic see separation such as separation via capillary electrophoresis (CE), liquid chromatography (LC) or high pressure liquid chromatography (HPLC), since 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 out of a capillary under a pressure of up to 4 bar, advantageously up to 2 bar, which encloses the inlet capillary of the eluent. In principle, higher pressures are also possible. In the preceding chromatographic separation, so-called normal phase (e.g. silica gel, aluminum oxide, aminodeoxyhexite, 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 Cg or Cis 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 and / or ammonium ions). Another advantage is that multiply charged ions can also be detected, so that ions with a molecular weight of up to a hundred thousand daltons can be detected. In the method according to the invention, molecular weights in a range from 1 to 10,000 daltons, preferably in a range, can advantageously be detected from 50 to 8000 daltons, particularly preferably in a range from 100 to 4000 daltons. Ion spray ionization, atmospheric pressure ionization (APCI) or thermoset ionization may be mentioned as further exemplary methods.

In the above-mentioned ionization methods, the ionization process takes place under atmospheric pressure and is essentially divided into three phases: First, the solution to be analyzed is in a strong electrostatic field, which is advantageous by generating a potential difference of 2-10 kV, 2-6 kV , is generated between the inlet capillary and a counter electrode. An electrical field between the inlet capillary tip and the mass spectrometer penetrates the analyte solution and separates the ions in an electrical field. Positive ions are drawn to the surface of the liquid in the so-called positive mode, negative ions in the opposite direction or vice versa in measurements in the so-called positive mode. The positive ions accumulated on the surface are then pulled further towards the cathode. When using spray capsules (NanoSpray) in which the If the solution seeking is not pressed out of the capillary by applying pressure, a liquid cone, the so-called Taylor cone, is formed, 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 flow at its syringe. The Taylor cone is not as pronounced when pressure-assisted spraying of the solution to be examined (eg with HPLC).

An aerosol is formed, which consists of analyte and solvent. The desolvation of the drops formed takes place in the following stage, which leads to the successive reduction in the drop size. The evaporation of the solvent is controlled by thermal action, e.g. by supplying hot inert gas. Evaporation in conjunction with the electrostatic forces increases the charge density on the surface of the sprayed substance mixture droplets. If the charge density or its charge repulsion forces then exceeds the surface tension of the droplets (so-called Raleigh limit), these droplets explode (Coulomb explosion) into smaller partial droplets. This process "solvent evaporation / Coulomb explosion" is repeated several times until the ions finally pass into the gas phase. To good ones

In order to obtain measurement results, the gas flow in the interface, the heating temperature applied, the flow rate of the heating gas, the pressure of the nebulizing gas and the capillary voltage must be precisely monitored and controlled.

The different ionization processes can be used to generate single or multiple charged ions. For the process according to the invention, processes for atomizing the substance mixture in the electric field, such as the thermospray, the electrospray (= ES) or the atmospheric pressure chemical ionization (= APCI) process, are advantageously used as the ionization process. With APCI ionization, the ionization takes place in a so-called coronna discharge. The thermospray or electrospray method is preferred, and the electrospray method is particularly preferred. The ionization space is connected to the following mass spectrometer via an interface, that is to say via a micro-opening (100 μm). On the side of the ionization chamber there is an interface plate with a larger opening. A heated carrier gas (= curtain gas), for example nitrogen, is blown in between this plate and the so-called "orifice". The nitrogen collides with the ions generated, for example, by electrospray, which were generated in the substance mixture. the. Blowing in the curtain gas advantageously prevents neutral particles from being sucked into the high vacuum of the subsequent mass spectrometer. Furthermore, the desolvation of the ions is supported by the curtain gas.

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 Paul et al. a first such device. These devices have an advantageous mass range up to approximately m / z = 4000 and achieve resolution values between 500 and approximately 5000. They have a high ion transmission from the source to the detector, are easy to focus and calibrate and advantageously have a high stability of the Calibration in continuous operation. Triple quadrupole instruments are the standard instruments for low-energy collision activation studies. These devices usually 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 high vacuum (approx. 10 ^{~ 5} Torr), the mass ( n) 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 generally used to focus the ions. Instead of this or these upstream quadrupoles, so-called "cones" lenses or lens systems can also be used to focus and introduce the ions into the first analytical quadrupole. Combinations of quadrupoles and cones can also be implemented and used.

Another quadrupole following Q1 (= II or Q2) serves as a collision chamber. The ions are advantageously fragmented in it 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 with a collision gas such as a noble gas such as argon or helium or another gas such as CO or nitrogen or mixtures of these gases such as argon / helium or argon / nitrogen for the fragmentation in the process according to the invention. Argon and / or nitrogen is preferred for reasons of cost. In the collision chamber, the collision gas is present in the process according to the invention with a pressure of 1 × 10 ^-5 to 1 × 10 ^-1 Torr, preferably 10 ^{~ 2} . Nitrogen is particularly preferred. Even without the application of a fragmentation voltage, the ions in the collision chamber may be fragmented in the presence of a collision gas. Between the qua- drupol Q1 and Q2 may have additional quadrupoles or cones for guiding the ions.

Finally, another quadrupole (= III or Q3) connects to the quadrupole Q2, which serves as a collision chamber. In this Q3, either the m / z quotients of individually selected fragments, several or all of the m / z quotients present in the substance mixtures after ionization (for the sake of simplicity referred to as mass or masses in this application) can be determined 10. Additional quadrupoles or cones can also be present between the quadrupole Q2 and Q3 to direct the ions.

In the method according to the invention, individual quadrupoles can be used

Collection of ions can also be operated as ion traps

15 after which the ions are then released for analysis again.

The quadrupoles used in the triple quadrupole mass spectrometers generate a three-dimensional electric field in the

20 the ions generated can be held or directed. They usually consist of 4, 6 or 8 rods or rods with the help of which an oscillating electric field is generated, with opposite rods being electrically connected. In addition to the designation quadrupole, the designations hexa-

25 or Octapol used. In the present application, these terms are intended to be included when the term quadrupole is used. Advantageous in the quadrupoles of the triple quadrupole mass spectrometer for guiding the ions are only low acceleration voltages of a few volts, preferably a few

30 10 V required.

Mixtures of substances such as animal or vegetable extracts, preferably vegetable extracts, are advantageously used in the process according to the invention. 35

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

I) In process steps (a) to (c), the mass of at least one ion present in the substance mixture after ionization in Ql 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 fragment ions formed is identified in a further analytical quadrupole Q3 and advantageously also quantified. The fragment ion to be analyzed is selected in such a way that this ion is advantageous has a high intensity, an easily identifiable characteristic mass and, in an advantageous embodiment of the method, enables easy quantification.

II) Then, in process step (d), the masses of all ions present in the substance mixture after ionization are analyzed, the quadrupole Q2 used as the collision chamber being always filled with collision gas, but in process step (d) no fragmentation voltage being present at Q2. In principle, this analysis can be carried out both with Q1 and with Q3, but it is more advantageous to carry out the analysis with Q3, since Q2 lies between Q1 and the quadrupole Q2 used as a collision chamber as a collision chamber. If a fragmentation occurs in Q2 despite the absence of a fragmentation voltage, this has no influence on a possible detection of the ion masses at the detector. In the case of a mass analysis with Q1, such a question in Q2 would lead to incorrect conclusions in the detection. Mass detection with Q3 is therefore preferred because possible sources of error are eliminated or are negligible.

The process steps (I) and (II) listed above can also be carried out in reverse order. FIG. 1 shows the sequence of the method according to the invention. In the process according to the invention, process steps (b) to (d) and (e) are advantageously carried out at least once within 0.1 to 10 seconds, preferably at least once within 0.2 to 6 seconds, particularly preferably within 0.2 to 2 Seconds, very particularly 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 carried out two to three times, preferably three times, within 0.2 to 6 seconds. The quadrupole Q2, which acts as a collision chamber, is constantly filled with collision gas in order to enable measurements of this type which follow one another rapidly and quickly. As our own measurements showed, this has no negative impact on the reproducibility of the measurements.

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

With the aid of the method according to the invention, in addition to the analysis of all the masses present in a substance mixture, individual substances or their masses can also be advantageously analyzed and quantified.

In principle, cleaning of the substance mixtures is not necessary in the process according to the invention. The substance mixtures can be measured immediately after being introduced into an ion source. This also applies to complex substance mixtures. Also, as internal standards, no marked or unlabeled pure substances of possible substances contained in the mixtures have to be added to the substance mixtures, although this is of course possible and subsequent quantification of the substances contained in the mixtures is simplified.

Purification by methods known to those skilled in the art, such as chromatographic methods, is, however, advantageous. On account of the ionization method preferred in the process according to the invention by atomizing the substance mixtures in the electric field, a purification and / or pre-purification of the substance mixtures can be coupled very easily to the mass spectrometric analysis, for example by means of chromatography. All separation methods known to the person skilled in the art, such as LC, HPLC or capillary electrophoresis, can be used as the chromatographic method. Separation methods based on adsorption, gel permeation, ion pair, ion exchange, exclusion, affinity, normal phase or reversed phase chromatography, to name just a few, can be used. Chromatographs based on normal phase and / or reversed phase, preferably reversed-phase columns with different hydrophobic modified materials such as C ₄ -, Cs ~ or Cιs ~ phases are advantageously used.

In the method according to the invention, a coupling of purification methods is advantageous from chromatography methods with a flow rate of the eluent (analyte + solvent) advantageously between 1 μl / min to 2000 μl / min, preferably between 5 μl / min to 600 μl / min, particularly preferably between 10 μl / min up to 500 μl / min possible, for example. Lower or higher flow rates can also be used without difficulty in the process according to the invention.

In principle, all protic or aprotic polar or non-polar solvents can be used as solvents for the purification process, which can be used with the subsequent analysis. are compatible. The person skilled in the art can easily determine whether a solvent is compatible with mass spectrometry by simple stitch tests. Suitable solvents are, for example, solvents that carry little or no charges, such as aprotic apolar solvents, which have a low dielectric constant (E _τ <15), low dipole moments (μ <2.5D) and low E _T ^N values (0.0 - 0.5) are characterized. Dipolar organic solvents or mixtures thereof are also suitable as solvents for the process according to the invention. Examples of suitable solvents are methanol, ethanol, acetonitrile, ether and heptane. Weak acidic solvents such as 0.01-0.1% formic acid, acetic acid or trifluoroacetic acid are also suitable. Weakly basic solvents such as 0.01-0.1% 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. Mixtures of the abovementioned solvents are also advantageous. The buffers customary in biochemistry are also suitable as solvents, advantageously buffers <200 mM, preferably <100 mM, particularly preferably <50 mM, very particularly preferably <20 mM. It is also advantageous, if buffers> 100 mM are used for the preparation of the substance mixture, that the buffers are completely or partially removed, for example by dialysis. Acetate, formate, phosphate, tris, MOPS, HEPES or mixtures thereof may be mentioned as buffers. High buffers and / or salt concentrations negatively influence the ionization processes and should be avoided if necessary.

In the process according to the invention, molecules which are contained in the substance mixtures can be from 100 Dalton (= D) to 100 Kilodalton (= kD), preferably from 100 D to 20 kD, particularly preferably from 100 D to 10 kD, very particularly preferably from Detect 100 D to 2000 D, ie identify and if necessary also quantify.

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

In the method according to the invention for analyzing the 5 substance mixtures, an internal standard such as e.g. Peptides, amino acids, coenzymes, sugars, alcohols, conjugated alkenes, organic acids or bases are added. This internal standard advantageously enables the quantification of the compounds in the mixture. In this way, substances contained in the substance mixture can be more easily analyzed and ultimately quantified.

Labeled substances are advantageously used as the internal standard, but in principle non-labeled substances are also suitable as the internal standard. Such similar chemical compounds

15 bonds are, for example, so-called compounds of a homologous series, the members of which differ only by, for example, an additional methylene group. As an internal standard, at least one isotope is preferably selected from the group ² H, "c, ¹⁵ _N ," 0, ¹⁸ 0, ³³ S, ³ S, ³⁶ S, ³⁵ cl, ³⁷ C1, ² S ±,

20 ³⁰ Si, ⁷ Se or mixtures thereof labeled substances used. ² H or ¹³ C is preferably used as the isotope for reasons of cost and for reasons of accessibility. These internal standards do not need to be complete for analysis, that is, they must be fully marked. A partial marking is completely sufficient. Advantageous

In the case of a marked internal standard, a substance is selected that has the highest possible homology 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 knife

30 results and the more precisely the connection can be quantified.

It is advantageous for the method according to the invention and especially for the quantification of the substances present in the mixture.

35 is liable to use the internal standard in a favorable relationship to the substance to be measured. Ratios of analyte (= connection to be determined) to internal standard greater than 1:15 do not improve the measurement results, but are possible in principle. A ratio of analyte to is advantageous

40 internal standard 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 method according to the invention can be prepared manually or advantageously automatically using conventional laboratory robots. Analysis with the mass spectrometer after any chromatographic separation can also be carried out manually or advantageously be carried out automatically. By automating the method according to the invention, mass spectrometry can advantageously be used for the rapid screening of various substance mixtures, for example plant extracts, in what is known as high-throughput screening. The method according to the invention is characterized by high sensitivity, good quantifiability, excellent reproducibility, and the lowest sample consumption. With the method, mixtures of biological origin, for example, new mutants of known or unknown enzymatic activities can be quickly obtained after a mutagenesis, for example after a classic 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 the so-called gene shuffling.

The method according to the invention enables the analysis of a wide 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, and a high scanning speed both in the fill scan mode of all substances in the substance mixtures and also in multiple reaction monitoring mode [= MRM, process steps (a) to (c)]. Furthermore, the method has a very high sensitivity and excellent calibration stability. Furthermore, it is excellently suited for continuous operation and therefore for use in HTS screening.

The invention is illustrated by the following examples:

Examples

1. Examples of MRM + FS measurements

a) TIC of the MRM + FS measurement

FIG. 2 shows the total ion chromatogram of an MRM + fill scan measurement [MRM = multiple reaction monitoring, FS = fill scan, TIC = total ion chromatogram, XIT = sum of several 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 a suitable solvent.

The representation of the measurement selected in FIG. 2 shows the summation of the intensities (y-axis) measured on the detector at the respective times (x-axis) from the two mass spectrometric experiments of multiple reaction monitoring (MRM). and the fill scan (FS). The chromatogram in FIG. 2 thus represents the sum of the TIC chromatograms of the two aforementioned mass spectrometric experiments.

b) TIC of the MRM experiment and TIC of the FS experiment

FIG. 3 shows the total ion chromatogram of the MRM experiment from an MRM + FS measurement.

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

c) TIC of the FS experiment

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

FIG. 6 shows the TIC of the FS experiment. The summation of all FS mass spectra, which were recorded in the hatched time window, are shown in FIG. 7.

d) TIC of an MRM experiment

A total ion chromatogram of an MRM + fill scan measurement is shown in FIG. 8 as in FIG. A calibration sample was measured.

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

FIG. 9 shows an extracted chromatogram in which coenzyme Q 10 was identified.

FIG. 10 and FIG. 11 show the identification of capsanthin and bixin, respectively.

FIG. 12 shows a total ion chromatogram of a filling scan of a plant extract. FIGS. 13 to 15 show the masses of different analytes in the extracted chromatogram, the assignment of which to a specific structure still has to be made.

To date, 200 further analytes have been selectively detectable in the described method.

Claims

claims

1. Mass spectrometric method for the analysis of substance mixtures with a triple quadrupole mass spectrometer, the substance mixtures being ionized before the analysis, characterized in that the method comprises the following steps:

a) selecting a mass / charge quotient (m / z) of an ion formed by ionization in a first analytical quadrupole (I) of the mass spectrometer,

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

d) Analyzing the mass / charge quotients of all im

Substance mixture of ions present through 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) can also be carried out in the reverse order. , A method according to claim 1, characterized in that the ionization of the substance mixture is chromatographic

Separation is upstream. , Method according to claim 1 or 2, characterized in that the chromatographic separation is an HPLC separation. Method according to claims 1 to 3, characterized in that steps (a) to (d) are carried out at least once within 0.1 to 10 seconds.

Method according to claims 1 to 4, characterized in that steps (a) to (d) are carried out at least once within 0.2 to 2 seconds.

Process according to claims 1 to 5, characterized in that the ionization takes place by evaporation of the substance mixture and ionization in the gas phase, by desorption of the substance mixture on a surface or by atomization of the substance mixture in an electric field.

Method according to claims 1 to 6, characterized in that the ionization is carried out by atomizing the mixture of substances in an electric field.

Method according to claims 1 to 7, characterized in that between 1 and 100 mass / charge quotients of various ions formed and selected by ionization are analyzed in step (a).

Method according to claims 1 to 8, characterized in that the mixture of substances biological or chemical

Is of origin.

Method according to claims 1 to 9, characterized in that the substance mixtures are derivatized according to claim 2 or 3 before the analysis or before the chromatographic separation.

Method according to claims 1 to 10, characterized in that the method is carried out manually or automatically.

Method according to claims 1 to 11, characterized in that the method is used in a high throughput screening.

A method according to claims 1 to 12, characterized in that the fragment ion analyzed in step (c) and the (m / z) quotients of all ions present in the substance mixture analyzed in step (d) or that analyzed in step (c) Fragment ion or those analyzed in step (d)

(m / z) quotients of all ions present in the substance mixture are quantified.