NL2026049B1

NL2026049B1 - Quantitative detection of reducing sugars

Info

Publication number: NL2026049B1
Application number: NL2026049A
Authority: NL
Inventors: Kleinnijenhuis Anne
Original assignee: Ducares B V
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2022-03-15
Also published as: WO2022015162A1

Abstract

The invention is directed to a method for quantitative detection of reducing sugars that are labeled With an amine-comprising labeling agent by using reducing sugars labeled With a 13C-isotope of said amine-comprising labeling agent as internal standards. In a further aspect, the invention is directed to 13C-isotope of a labeling agent selected from the group consisting of 2- aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB) and other labeling agents.

Description

P127573NL00 Title: Quantitative detection of reducing sugars The invention relates to the quantitative detection of reducing sugars in a sample.

In particular, the invention relates to the quantitative detection of reducing sugars using corresponding '3C-labeled reducing sugars as internal standards in chromatography - mass spectrometry coupled analyses.

Reducing sugars such as reducing oligosaccharides are frequently used as food supplements.

For example, human milk oligosaccharides (also referred to as HMOs) are known for positive effects on infants and can therefore be added to infant milk formula.

For the purpose of quality control of the infant milk formula or other mixtures containing reducing sugars, it is desired to determine the amount of these reducing sugars in the mixtures.

Quantitative detection of reducing sugars can conventionally be carried out using liquid chromatography coupled to mass spectrometry (also referred to as LC-MS) using external standards.

These methods are based on analyzing a sample containing a known amount of the reducing sugars (i.e. the external standard), followed by analyzing an analyte sample containing an unknown amount of the reducing sugars and comparing the response for the reducing sugars in the analyte sample with that of the external standard.

A drawback of this approach is however the inherent variations in responses per analysis and per component.

These response variations may for instance, amongst other causes, be due to differences in the environment wherein the external standard and the reducing sugars in the analyte sample are analyzed, the so-called matrix effects.

Additionally, the status of the LC-MS system generally affects the response.

The response variations result in undesirable inaccuracies of the analytical method and can even result in false negative results.

Response variations can be corrected by using an internal standard in the analyte sample.

In Yuan et al.

Journal of Chromatography

A, 1067 (2005) 145-152, analyte monosaccharides were labeled with 2- aminopyridine and then separated and monitored by LC-MS, using tetradeuterium-labeled pyridylamino monosaccharides as internal standards. Labeling or tagging is a technique known to facilitate detection after chromatographic separation and is occasionally used for glycomics analysis (see also Ruhaak et al., Analytical and Bioanalytical Chemistry 397 (2010) 3457-3481). A drawback of labeling the internal standard with a deuterated label, is that the deuterium atoms in the labels lead to a slight change in polarity and to retention time shifts in LC of the internal standards vis-a-vis the analytes (see also Iyer et al., Journal of Chromatographic Science 42 (2004) 383-387). The non-labeled and labeled equivalents will then elute in different environments, especially in complex samples.

The object of the present invention is to provide an improved method for the quantitative detection of reducing sugars by chromatography/mass spectrometry.

Surprisingly, the present inventor has found that this object can be met by providing a method for quantitative detection of reducing sugars that are labeled with an amine-comprising labeling agent by using reducing sugars labeled with a 13C-isotope of said amine-comprising labeling agent as internal standards.

Accordingly, the present invention is directed to a method for the quantitative detection of one or more reducing sugars in a sample comprising: - providing said reducing sugars labeled with an amine- comprising labeling agent; - providing a known amount of reducing sugars labeled with an isotopic amine-comprising labeling agent, wherein said isotopic amine- comprising labeling agent is a 13C-isotope of said amine-comprising labeling agent;

- providing a sample mixture comprising said reducing sugars labeled with the amine-comprising labeling agent and the reducing sugars labeled with the isotopic amine-comprising labeling agent; - analyzing the sample mixture with chromatography coupled with mass spectrometry and determining the amount the reducing sugars labeled with the amine-comprising labeling agent in the sample mixture using the reducing sugars labeled with the isotopic amine-comprising labeling agent as internal standards.

The labeling of reducing sugars with amine-containing labeling agents (herein also referred to as labeling agents) can be based on a reductive amination reaction of the reducing sugars with the labeling agents. In Ruhaak et al., Analytical and Bioanalytical Chemistry 397 (2010) 3457-3481), various labeling strategies and suitable reducing reagents are described. Advantageously, the reductive amination can be carried out essentially quantitatively, meaning that essentially all reducing sugars molecules in the sample to be analyzed are labeled. The determined amount of the labeled reducing sugars therefore typically directly corresponds to the amount of reducing sugars in the sample to be analyzed.

The labeling agent can in principle be any type of amine- containing labeling agent that can be reacted quantitively with the reducing sugars in a reductive amination reaction. Labeling agents that comprise a fluorescent moiety are particularly preferred, as these enable UV-VIS and fluorescence detection of the labeled reducing sugars in the chromatography. Labeling agents that are conventionally known for fluorescence detection of reducing sugars (see for instance Anumula, Analytical Biochemistry 350 (2006) 1-23) and that are suitable for the present invention include 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP), 2-aminoacridone (AMAC), 3-(acetylamino)- 6-aminoacridin (AA-Ac), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), 8-aminopyrene-1,3,6- trisulfonic acid (APTS), aminoquinoline (6-AQ), 7-

aminomethyl-coumarin (AMC), 2-amino(6-amido-biotinyl) pyridine (BAP), 9- fluorenylmethoxycarbonyl (FMOC)-hydrazide, 1,2-diamino-4,5- methylenedioxy-benzene (DMB), o-phenylenediamine (OPD).

The labeling agents 2-AB, 2-AA, 2-PA, ANTS and APTS are particularly preferred. 2-AB is most preferred for its particular good performance in LC-MS.

The isotopic amine-comprising labeling agent (herein also referred to as isotopic labeling agent) that is used according to the present invention, is a 13C-1sotope of said amine-comprising labeling agent. As such, in the chromatography, the retention time of the reducing sugars labeled with the labeling agents (herein also referred to as labeled reducing sugars) is the same as the retention time of the reducing sugars labeled with the isotopic labeling agent (herein also referred to as isotopic labeled reducing sugars or internal standard). The labeled reducing sugars can be distinguished from the isotopic labeled reducing sugars in the mass spectrometer.

To facilitate differentiation between the labeled reducing sugars and the isotopic labeled reducing sugars in mass spectrometry, it is preferred that the isotopic labeling agent comprises four or more 13C atom. In the sample to be analyzed, naturally occurring isotopes of the labeled reducing sugars will be present and the exact mass of each of these will give a corresponding signal on the mass spectrum obtained in the mass spectrometer. This leads to a peak pattern in the spectrum corresponding to each labeled reducing sugar. To minimize or prevent overlap of the peak patterns of the labeled reducing sugars with the isotopically labeled reducing sugars, it is preferred that the nominal masses of the labeled reducing sugars and the isotopic labeled reducing sugars differ with more than 4 u. Accordingly, it is preferred that the isotopic labeling agent comprises more than 4, more preferably 6 13C-atoms.

In order to minimize a difference in retention time of the labeled reducing sugars and the isotopic labeled reducing sugars and/or to facilitate the preparation of the isotopic labeling agents, it 1s preferred that the carbon atoms in the isotopic labeling agent being 1°C-atoms are present in an aromatic moiety of the isotopic labeling agent. Most of the fluorescent labeling agents comprise such an aromatic moiety. For instance, for 2-AB 5 and 2-AA, it is preferred that the benzene ring of these compounds comprises more than 2, preferably more than 4, most preferably 6 13C- atoms.

The reducing sugar according to the present invention may be any type of compound comprising a reducing sugar moiety that is capable of acting as a reducing agent because it has a free aldehyde group or a free ketone group. Accordingly, the reducing sugar may be a monosaccharide, an oligosaccharide or a compound containing one or more sugar groups. In principle, all monosaccharides are reducing sugars, along with some disaccharides, some oligosaccharides, and some polysaccharides. The monosaccharides can be divided into two groups: the aldoses, which have an aldehyde group, and the ketoses, which have a ketone group. Ketoses, which are also considered reducing sugars according to the present invention, preferably first tautomerize to aldoses before they can be labeled with the labeling agent. The common dietary monosaccharides galactose, glucose and fructose are all reducing sugars.

The present invention is suitable for the quantitative determination of reducing oligosaccharides, particularly human milk oligosaccharides present in infant milk formula. Accordingly, in a preferred embodiment of the present invention the sample to be analyzed comprising the one or more reducing sugars is an infant milk formula sample. The method may therefore further comprise providing an infant milk formula comprising the reducing sugars and labeling said reducing sugars to provide the reducing sugars labeled with the amine-comprising labeling agent.

There are many human milk oligosaccharides (HMOs) known, see for example Chen, "Human Milk Oligosaccharides (HMOs): Structure,

Function, and Enzyme-Catalyzed Synthesis", Advances in Carbohydrate Chemistry and Biochemistry 72 (2010) 113-190. One particular advantage of the present invention is the flexibility towards the reducing sugars that can be analyzed. More specifically, the labeling of the reducing sugars with the isotopic labeling agent allows quick and predictable 13C-labeling in case a novel analyte is to be analyzed and a novel internal standard must be provided. In other words, the internal standard can be prepared with standard reduction amination and it 15 not required to provide a new and complex synthetic pathway to prepare a 1*C-isotope of the novel reducing sugar itself. Thus, although the present invention is particularly suitable for the HMOs specifically mentioned and exemplified herein, the invention is not limited to those HMOs. If new HMOs are found and/or if additional HMOs are added to certain infant milk powders, the analytical method according to the present invention can be easily expanded.

Advantageously, the present method enables the separation and quantitative detection of more than two HMOs in the sample. Accordingly, in a preferred embodiment, the sample to be analyzed comprises more than 2, preferably more than 4, most preferably more than 6 different types of reducing sugars, preferably HMOs.

The present invention was found to be particularly suitable for the quantitative detection of 2’-fucosyllactose (2'FL), 3’-fucosyllactose (3’FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3’SL) and 6’-sialyllactose (6’SL). Other candidates include lactose, HMOs containing 3 to 8 sugar units and any other HMO as they all contain lactose at their reducing end, which is reaction site of the labeling agent. In a particularly preferred embodiment, the sample to be analyzed comprises at least 4, preferably at least 6, most preferably all of these HMOs and the method comprises the quantitative detection of all of these HMOs present in the sample.

The sample to be analyzed may contain more than one reducing sugar. Moreover, the sample may contain more components than only reducing sugars. For example, in case the sample is an infant milk formula sample, it may further contain proteins, fats, vitamins and the like. In certain embodiment, fat may be removed from the sample before it is subjected to the chromatography step and an internal standard may be added to correct for volume. In order to effectively separate the reducing sugars from other components, the sample mixture comprising the labeled reducing sugars, the isotopic labeled reducing sugars and possible other component is separated by chromatography coupled with mass spectrometry (herein also referred to as chromatography/mass spectrometry). In general, the sample mixture will be provided and separated as a liquid, in which case liquid chromatography (also referred to as LC) is applied. Ultra-performance liquid chromatography (UPLC) is particularly preferred.

Reducing sugars are typically hydrophilic and accordingly conventionally separated using hydrophilic interaction chromatography (HILIC) with a polar stationary phase. The present inventor found that the labeled reducing sugars according to the present invention can also be separated using HILIC, but that particular good results can be obtained when the labeled reducing sugars are separated using reversed phase chromatography (RPC). In RPC, a non-polar stationary phase is used.

Although it is preferred to separate all components in the sample mixture by chromatography as much as possible, in certain embodiments the labeled reducing sugars may not be fully separated from other labeled reducing sugars, or other components Gf present) in the sample mixture. This is not necessarily detrimental to the result of the method, as a further separation can be carried out by mass spectrometry, which may be preceded by separation using 10n mobility.

The mass spectrometry preferably comprises at least two mass analyzers (MS/MS), e.g. which can be achieved in a triple quadrupole mass spectrometer (QqQ) or a quadrupole - time of flight mass spectrometer (q- ToF). Other suitable mass analyzers include Orbitrap, 10n trap or Fourier Transform Ion Cyclotron Resonance (FTICR). In a preferred embodiment, the mass spectrometry comprises a fragmentation method, e.g. collision induced dissociation (CID), to further improve the separation capability of the analysis.

Preferably, the peak area in the extracted chromatogram is used for determining the amount of the labeled reducing sugars in the sample mixture. Since the isotopic labeled reducing sugars have essentially the same retention time in the chromatography, the isotopic labeled reducing sugars can directly be used as internal standards to the corresponding labeled reducing sugars.

In another aspect, the invention is directed to the isotopic labeling agents. More specifically, the invention is further directed to a 15C-isotope of alabeling agent selected from the group consisting of 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP), 2-aminoacridone (AMAC), 3-(acetylamino)-6-aminoacridin (AA-Ac), 8-aminonaphthalene- 1,3,6-trisulfonic acid (ANTS), 8-aminopyrene-1,3,6- trisulfonic acid (APTS), aminoquinoline (6-AQ), 7-aminomethyl-coumarin (AMC), 2-amino(6-amido- biotinyl) pyridine (BAP), 9-fluorenylmethoxycarbonyl (FMOC)-hydrazide, 1,2-diamino-4,5-methylenedioxy-benzene (DMB), o-phenylenediamine (OPD), preferably 2-AB, 2-AA, 2-PA, ANTS and APTS, most preferably 2- AB.

Carbon-13 (i.e. the carbon atom with an atomic mass of about 13 u, also referred to as 13C) is a stable isotope of carbon. The natural occurrence of 1°C is about 1.109%, based on all carbon atoms. The isotopic labeling agent according to the present invention is enriched in !5C, meaning that the amount of !5C of specified carbon atoms in the agent is higher than the natural occurrence. Although in principle, the enrichment in C is generally essentially 100 atom®%, in practice, the enrichment m 15C may be below 100 atom%, meaning that less than 100% of the atoms that are specified to be 15C are actually 15C. Typically, the isotopic labeling agent is 13C-enriched for at least 90 atom%, preferably for at least 95 atom%, most preferably at least 99 atom%. If an isotopic labeling agent is used in the present invention with an 3C-enrichment of slightly less than 100%, it is generally preferred to correct the signal response obtained in the mass spectrometry for this 13C-enrichment.

The isotopic labeling agent can be prepared with 1C-enriched starting materials, such as for example 99 atom% *C aniline. This means that about 99% of the specified carbon atoms in an aniline sample are 13C atoms. For example, in 99 atom % aniline-13Cg, at least 99% of all carbon atom in the aniline sample are 15C atoms, or in other words, on average at least 99% of all specified 13C atoms are actually 13C atoms. The 13C- enrichment of a isotopic compound can be determined with mass spectrometry.

The isotopic labeling agent in accordance with the present invention preferably comprises more than 2, preferably more than 4, most preferably at least 6 3C-atoms. In the embodiments wherein the isotopic labeling agent comprises 2-AB or 2-AA, the benzene ring of these agents preferably comprises more than 2, preferably more than 4, most preferably 6 13C-atoms.

Yet another aspect of the invention is directed to the preparation of the isotopic labeling agent, specifically to the preparation of 2-AB and/or 2-AA.

A synthetic pathway for the preparation starting from aniline with formula (I) is shown in Scheme 1.

* * OH, * X * Nog Ol Ho NO Step a Ol C Step b NH, + d , “NH 0 , 0) ah (IN . 0 i © © Ce Step c ry Step d Oh TN — YN — TNF SNH, (IV) Vv) (VI) Scheme 1 In the compounds according to formulae (I)-(VI), at least one, preferably more than 2, more preferably more than 4, most preferably all of the carbon atoms labeled with an asterisk (*) is a 13C atom in accordance with the invention.

An embodiment of the present invention is accordingly directed to a method for the preparation of AB-2 and/or AA-2, i.e. the compound of formula wherein X is respectively NH: or OH, comprising step d of hydrolyzing the anhydride of formula (V) with ammonia or water, respectively.

In a preferred embodiment of the invention, the method further comprises step c of reacting the compound of formula (IV) (also referred to as isatin) in a Baeyer-Villiger oxidation, preferably using hydrogen peroxide, to obtain the anhydride of formula (V).

In a further preferred embodiment of the invention, the method further comprises step b of reacting the compound of formula (III) (also referred to as isonitroso acetanilide) with acid, preferably sulfuric acid, to obtain isatin.

In a most preferred embodiment of the invention, the method further comprises step a of reacting the compound of formula (I) (also referred to as aniline) with the compound of formula (I) (also referred to as chloral hydrate) to obtain isonitroso acetanilide. For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. The invention can be illustrated with the following non-limiting examples. Example 1 - preparation of 99 atom 13C6-2-AB

OH s Hos * \ cp Cl cl Ol L “OH H,S0, ee” ANH, H*,Na2804, ON 0 TN

1.2 H,O, NH,OH 1.5 1.4 The synthesis of 99 atom% 3Cg isonitroso acetanilide (1.5) was performed using aniline-!5C (1.0 g, 1.0 eq.), chloral hydrate (1.1 eq.), sodium sulfate (9.8 eq.), hydrogen chloride (0.8 eq.) and hydroxylamine hydrochloride (3.2 eq.) in water at reflux temperature. The concentration of the reaction mixture was increased with a factor 3. Cooling to room temperature resulted in precipitation of the product. Work-up of the reaction mixture provided isonitroso acetanilide (1.5) in quantitative yield and an acceptable purity for further synthesis according to ‘H-NMR and HPLC-MS analysis. The synthesis of isatin 1.4 was performed using compound 1.5 (~

1.7 g) in neat sulfuric acid (18.5 eq.) at 80 °C. Twenty minutes of heating provided (almost) full conversion of compound 1.5. Work-up of the reaction mixture provided isatin 1.4 (0.71 g) in 46% yield. . DP on 9 > 14 “ Has Ne 1

Baeyer Villiger oxidation towards compound 1.3 was performed using isatin 1.4 (labeled)(0.36 g) and meta-chloroperoxybenzoic acid (mCPBA) in dichloromethane (DCM) at room temperature. Work-up of the reaction mixture provided crude compound 1.3 (labeled) in 96% yield with minor impurities present.

Hydrolysis of crude compound 1.3 (0.382 g) was performed using ammonium in water at room temperature. Analysis of the reaction mixture showed the target compound (I) and the presence of side products. The reaction mixture was directly purified by automated normal phased column chromatography. This provided ~ 30 mg of the product I with a relatively high purity (>95% according to a standard HPLC-MS method). Further purifications of remaining crude batches yielded about 125 mg of additional product I.

Example 2 — preparation of internal standards with 2-AB and HMO The isotopic 2-AB (1 mg) prepared according Example 1 was mixed with 2-picoline-borane (2-PicBH3, 1.5 mg), methanol (126 pL) and acetic acid (14 pL). Then, an aqueous solution of the target HMOs (140 pL of 5ug/mL per HMO) was added and the resulting mixture was incubated at 65 °C for 2 hours. Then, 280 uL water was added to obtain the desired internal standard mix.

Example 3 — analysis of sample containing possible reducing sugars 2’FL, 3’FL, DFL, LNT, LNnT, 3’SL and 6’SL.

A milk powder sample (5 g) was dissolved in water (30 mL). The resulting sample was 100x diluted in water. The phase-separated fat layer was removed leaving an aqueous layer.

From the sample, an aliquot (200 pL) was transferred to a filter (cutoff 10 kDa) and water (200 pL) was added. The sample was then centrifuged for 30 minutes at 21000 ref.

An aliquot of the resulting ultrafiltrate (50 uL) was taken and mixed with the reagent (50 pL.) containing 2-AB (2.1 mg), 2-PicBH3, (3.2 mg), methanol (45 uL) and acetic acid (5 nL). The resulting mixture was incubated at 65 °C for 2 hours.

Then, 300 uL water was added and 100 pL of the resulting sample was transferred to a LC vial together with 10 pL of the relevant internal standards that were prepared according to Example 2. The final sample was run over a UPLC/MS.

In Figure

1 the combined extracted chromatogram is shown for an infant milk formula sample which contained 2’FL. and LNnT.

In Figure 2 the corresponding combined extracted chromatogram of the internal standard signals is shown.

Claims

Conclusions

A method for the quantitative detection of one or more reducing sugars in a sample to be analysed, said method comprising: - providing said reducing sugars labeled with an amine-comprising labeling agent; - providing a known amount of reducing sugars labeled with an isotopic amine-comprising labeling agent, wherein said isotopic amine-comprising labeling agent is a 15C isotope of said amine-comprising labeling agent; - providing a sample mixture comprising said reducing sugars labeled with the amine-comprising labeling agent and the reducing sugars labeled with the isotopic amine-comprising labeling agent; - analyzing the sample mixture by chromatography coupled to mass spectrometry and determining the amount of the reducing sugars labeled with the amine-comprising labeling agent in the sample mixture using the reducing sugars labeled with the isotopic amine-comprising labeling agent as internal standards.

Method according to the preceding claim, wherein the sample to be analyzed comprises at least 2, preferably at least 4, most preferably at least 6 different types of reducing sugars and wherein the amount of at least 2, preferably at least 4, most preferably at least 6 different reducing sugars labeled with the amine-comprising labeling agent in the sample mixture is determined.

A method according to any one of the preceding claims, wherein the reducing sugars comprise reducing oligosaccharides, preferably human milk oligosaccharides.

A method according to any one of the preceding claims, wherein the reducing sugars comprise one or more human milk oligosaccharides (HMOs), preferably one or more lactose-based HMOs, more preferably HMOs comprising 3 to 8 sugar units, most preferably one or multiple HMOs selected from the group consisting of 2'-fucosyllactose (2'FL), 3' fucosyllactose (3'FL), difucosyllactose (DFD), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) 3'-sialyllactose (3''SL) and 6'-sialyllactose (6'SL).

A method according to any one of the preceding claims, wherein the sample to be analyzed is a baby milk formula sample, preferably wherein the method comprises providing a baby milk powder comprising the reducing sugars and labeling said reducing sugars to have the reducing sugars labeled with the amino acid. comprehensive labeling means.

A method according to any preceding claim, wherein said amine-comprising labeling agent comprises a fluorescent moiety.

The method of any preceding claim, wherein said amine-comprising labeling agent is selected from the group consisting of: 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP) , 2-aminoacridone (AMAC), 3-(acetylamino)-6-aminoacridine (AA-Ac), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), 8-aminopyrene-1,3,6-trisulfonic acid ( APTS), aminoquinoline (6-AQ), 7-aminomethyl-coumarin (AMC), 2-amino(6-amido-biotinylDpyridine (BAP), 9-fluorenyl-methoxycarbonyl (FMOC)-hydrazide, 1,2-diamino-4,5 -methylenedioxy-benzene (DMB) and o-phenylenediamine (OPD), preferably 2-AB, 2-AA, 2-PA, ANTS and APTS, most preferably 2-AB.

A method according to any one of the preceding claims, wherein the sample mixture is analyzed by liquid chromatography coupled with mass spectrometry (LC-MS), preferably ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS).

A method according to the preceding claim, wherein the liquid chromatography comprises hydrophilic interaction chromatography (HILIC) or reversed phase chromatography, preferably reversed phase chromatography (RPC).

A method according to any one of the preceding claims, wherein the labeled reducing sugars are obtainable by reductive amination of the reducing sugars with the labeling agent and the isotopic labeling agent, respectively.

A method according to any one of the preceding claims, comprising performing a reductive amination of the reducing sugars with the labeling agent and/or with the isotopic labeling agent to obtain the reducing sugars labeled with the labeling agent and/or the isotopic labeling agent, respectively.

12. 13C-isotope of a labeling agent selected from the group consisting of 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP), 2-aminoacridone (AMAC), 3- (acetylamino)-6-aminoacridine (AA-Ac), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), 8-aminopyrene-1,3,6-trisulfonic acid (APTS), aminoquinoline (6-AQ), 7-aminomethyl-coumarin (AMC), 2-amino(6-amido-biotinyl) pyridine (BAP), 9-fluorenylmethoxycarbonyl (FMOC)-hydrazide, 1,2-diamino-4,5-methylenedioxy-benzene (DMB) and o-phenylenediamine (OPD), preferably 2-AB, 2-AA, 2-PA, ANTS and APTS, most preferably 2-AB.

13. C-isotope of the labeling agent according to the preceding claim, comprising more than 2, preferably more than 4, most preferably at least 6 C-atoms.

13 C-isotope of the labeling agent according to any one of claims 12-13 comprising 2-aminobenzamide (2-AB) or 2-amimobenzoic acid (2-AA) of which the benzene ring is more than 2, preferably more than 4, most preferably 6 13 C atoms.