PL230479B1 - Method for simultaneous quantitative analysis of low-molecular compounds of the pathway of nitrogen oxide synthesis in the plasma, serum, solid tissues and water solutions - Google Patents

Description

Description of the invention

The subject of the invention is a method for the simultaneous quantitative analysis of low-molecular compounds of the nitric oxide synthesis pathway, i.e. dimethylamine (DMA), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-arginine and L-citrulline in plasma, serum, urine, solid tissues and aqueous solutions.

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase and is continuously produced by the process of asymmetric protein methylation. Its synthesis and degradation are actively regulated processes. Asymmetric dimethylarginine is recognized as an independent marker of endothelial dysfunction and atherosclerosis in humans. Increased levels of ADMA in the serum have been found in many diseases of the cardiovascular system (atherosclerosis, hypertension, chronic heart failure), as well as in diabetes and hypercholesterolaemia. Due to the important role of nitric oxide in the human body, ADMA is also tested as a potential marker of other disease entities. On the other hand, SDMA has been recognized as an excellent marker of kidney function. There is also growing evidence that SDMA is involved in inflammation and atherosclerosis. However, they need to be further checked as SDMA is considered only a weak indirect inhibitor of nitric oxide synthase.

DMA and L-citrulline, which are products of ADMA enzymatic hydrolysis by the enzyme dimethylarginine dimethylaminohydrolase, are an important point in the study of asymmetric metabolism of dimethylarginine. As a result of the autooxidation of NO with DMA, N-nitrosodimethylamine (NDMA) is formed - a nitrosated derivative of dimethylamine, which as a result of metabolic reactions converts guanine to 0 ^6- methylguanine, which preferentially evaporates with thymine. As a result, during DNA replication, G: C-> A: T transitions in daughter cells take place. This modification is considered an essential step in the carcinogenesis process.

For these reasons, determining the exact concentration of DMA, ADMA / SDMA, L-Arginine and L-citrulline along with their mutual correlations is an important element of diagnostic tests.

In the work of Luigi Servillo et al. (Luigi Servillo, Alfonso Giovane, Nunzia DOnoffio, Rosario Casale, Domenico Cautela, Domenico Castaldo, Maria Luisa Balestrieri, Int. J. Mol. Sci., 2013 Oct, 14, 20131-20138) the method of quantitative analysis of ADMA, SDMA and Arg bypassing the derivatization process using the HPLC-ESI-MS / MS technique. Due to the simultaneous quantitative analysis of monomethyl-L-arginine (NMMA), L-homoarginine (HArg) and L-arginine, it was necessary to obtain a complete chromatographic separation, which was possible only with a strictly defined composition of the mobile phase.

Michael J. Bishop et al. (Michael J. Bishop, Brian Crow, Dean Norton, Ekaterina Paliakov, Joe George, JA Bralley, Journal of Chromatography B, 859 (2007) 164-169), presented the method of quantitative analysis of Arg, ADMA and SDMA without the derivatization process, using microfiltration technique.

Edzard Schwedhelm et al. (Edzard Schwedhelm, Jing Tan-Andresen, Renke Maas, Ulrich Riederer, Friedrich Schulze, Rainer H. Boger, Clin Chem. 2005 Jul; 51 (7): 1268-71) presented the method of quantitative analysis of L-Arg, ADMA and SDMA in which all compounds were analyzed as derivatives of butyl esters.

From the publication of Jens Martens-Lobenhoffer et al. (Jens Martens-Lobenhoffer, Stefanie M. Bode-Bóger, Journal of Chromatography B, 798 (2003) 231-239) it is known to quantify Arg, ADMA, SDMA and L-citrulline in human plasma and urine using the HPLC-ESI technique -MS. The quantitative analysis of derivative compounds after the derivatization reaction with orthophthalic aldehyde (OPA) is presented. The possible use of OPA as a derivatizing compound has been proven in quantitative analysis using mass spectrometry. However, this method requires a chromatographic separation of ADMA from SDMA on an analytical column. The use of the same pseudomolecular ion for both derivatives of these compounds results in a significant extension of the analysis time (up to several dozen minutes).

In the work of Dimitrios Tsikas et al. (Dimitrios Tsikas, Bibiana Beckmann, Frank-Mathias Gutzki, Jens Jordan, Analytical Biochemistry, 413 (2011), 60-62) presents a method of simultaneous quantification of ADMA and SDMA in urine as pentafluoropropionic derivatives. This method allows simultaneous analysis of ADMA and SDMA using the GC-MS / MS technique, but requires long and laborious sample preparation.

Kristine Chobanyan et al. (Kristine Chobanyan, Anja Mitschke, Frank-Mathias Gut2ki, Dirk O. Stichtenoth, Dimitrios Tsikas, Journal of Chromatography B, 851 (2007) 240-249) and Dimitrios Tsikas et al. (Dimitrios Tsikas, Thomas Thum, Thomas Becker, Vu Vi Pham, Kristina Chobanyan, Anja Mitschke, Bibiana Beckmann, Frank-Mathias Gutzki, Johann Bauersachs, Dirk O. Stichtenoth, Journal

PL 230 479 Β1 of Chromatography B, 851 (2007) 229-239) presents the method of quantitative analysis of DMA with the use of its pentafluorobenzamide derivatives. This method enables rapid quantification of DMA in both plasma, serum and urine of test patients, using a minimum number of steps in sample preparation. The authors also tested the possibility of using the above-described method for the quantitative analysis of ADMA, but did not obtain suitable pentafluorobenzamide derivatives. This could be due to the use of an unsuitable organic solvent for the extraction / deproteinization of the samples.

The prior art needs to provide a fast and reliable method for the simultaneous quantification of low molecular weight compounds involved in the nitric oxide synthesis pathway, i.e. arginine, citrulline, ADMA, SDMA and DMA in plasma, serum, solid tissues and aqueous solutions. To date, no method has been provided in the prior art that would allow the efficient analysis of the above compounds simultaneously in one test.

The subject of the invention is a method of simultaneous determination of analytes selected from the group consisting of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-citrulline and dimethylamine (DMA) in plasma, serum, urine, and solid human tissues. or animal and aqueous solutions in the form of their pentafluorobenzamide derivatives, including the steps of:

A. the mixture of internal standards D6-DMA, D7-ADMA, D7-Arg is added to the samples of the tested material.

B. the pH of the reaction mixture is stabilized with Na2CO3 in the amount of 5-30 μΙ and in a concentration from 10 mM to 40 mM,

C. Acetonitrile (AcCN) is added to the mixture of reagents, the ratio of AcCN: H2O is from 3: 1 to 9: 1.

D. the analytes are derivatized through the -NH group using 2,3,4,5,6 pentafluorobenzoyl chloride added to the reaction mixture in the amount of 10 μΙ and a concentration of 5-30% with a reaction time of 5-10 minutes,

E. then the pentafluorobenzamide derivatives of the analytes are separated together with the internal standard with the appropriate separation technique.

Preferably, the mixture of the internal standards in (a) is D6-DMA (1 μΜ - 200 μΜ); D7-ADMA (4 μΜ -100 μΜ); D7-Arg (10 μΜ - 250 μΜ).

Preferably, the mixture of the internal standards in (a) is D6-DMA (50 µM); D7-ADMA (20 μΜ); D7-Arg (100 μΜ).

Preferably, the pH of the reaction mixture is stabilized with 10 μCO of Na2CO3.

Preferably, the concentration of AcCN is from 15-100%.

More preferably the concentration of AcCN is 100%.

Preferably, the AcCN: sample ratio is 5: 1.

Preferably, the concentration of 2,3,4,5,6 pentafluorobenzoyl chloride in acetonitrile is 10%.

Preferably, the reaction mixture formed in (d) after the addition of 2,3,4,5,6 pentafluorobenzoyl chloride is shaken for 3-10 min.

Preferably, the reaction mixture is shaken for 5 min.

Preferably, the reaction mixture is shaken at 25 ° C.

Preferably, the reaction mixture is shaken at 1100 rpm.

Preferably, the separation in (d) is performed by liquid chromatography using a detector-mass spectrometer or a tandem mass spectrometer operating in MRM mode or detectors such as LJV / VIS, DAD, FLD.

Preferably, the analysis is carried out in reverse phase with a concentration gradient of 0.1% formic acid in water and 0.1% formic acid in methanol.

Preferably, the separation in (d) is carried out by gas chromatography after removing residual water in the sample first.

The solution according to the invention, in which the reaction medium and at the same time the deproteinizing reagent is a mixture of acetonitrile and water, enables simultaneous derivatization of both dimethylamine and other compounds of the nitric oxide synthesis pathway and obtaining high sensitivity and specificity of the method. The reaction medium is acetonitrile, which simultaneously serves to deproteinize the sample in the case of a matrix such as, for example, plasma or serum. When there is no need to de-proteinate the sample, the AcCN concentration for aqueous samples is 100%.

The essence of the solution is to change the composition of the reaction mixture for the acylation of the amino group (Schotten-Baumann reaction), in which a two-phase system is usually used, where the basic

PL 230 479 Β1 water phase neutralizes the acid ammonium salt and the product remains in the organic phase. The use of basic acetonitrile with approx. 1/6 volume of water and an acylating agent - pentafluorobenzoyl chloride, allowed for efficient production of amide derivatives of the above-mentioned compounds. The two-phase system used by other researchers with e.g. water and nitrobenzene or water and toluene did not give such a possibility - it caused that more polar derivatives of amino acids went to the water phase, and less polar DMA derivatives to the organic phase.

The method does not require solid phase extraction or sample drying, which is often necessary for other procedures. The solvent used (acetonitrile) is relatively less harmful compared to e.g. benzene or nitrobenzene. Amide derivatives of the tested substances are very stable in time and separate well in the process of chromatography on a column with a reversed phase system, which facilitates subsequent analysis on a detector - mass spectrometer.

Simultaneous derivatization and extraction of the test sample is fast, accurate and precise, resulting in lower losses associated with both processes. An experienced analyst is able to prepare about 100 samples daily. Due to the important role of the tested compounds, it can be a useful technique in quantitative research in analytical laboratories.

The provided method of chromatographic separation, unlike many literature examples, does not require the use of properly prepared buffers as eluents. The use of 0.1% formic acid in water and 0.1% formic acid in methanol significantly simplifies the procedure and does not require special treatment of the chromatographic column as it is in the case of analyzes with the use of buffers. Reverse phase analysis with the use of the proposed concentration gradient also allows for the separation of ADMA and SDMA structural isomers, which is often a big problem.

The method of the invention allows for quick and simultaneous quantitative analysis of L-arginine, ADMA, SDMA, L-citrulline and DMA, both in plasma, serum, urine and solid tissues, using a minimum number of steps in the preparation of the sample. The method according to the invention is carried out using LC-MS, LC-MS / MS techniques.

The method according to the invention also allows the quantification of other primary and secondary amines present in biological matrices, including most amino acids and their derivatives. The pentafluorobenzamide derivatives formed in the reaction are stable when stored under appropriate conditions from - 80 ° C to a maximum of 10 ° C and allow for the quantitative analysis of the sought compounds with appropriate selectivity, sensitivity and linearity in a wide range of concentrations.

All LC-MS / MS measurements were performed in positive ionization mode and selective quantification mode - multiple reaction monitoring (MRM) observations. The fragmentation conditions of the selected molecules were optimized with the Optimizer Mass Hunter Workstation Software (Agilent Technologies).

Fig. 1 chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline and DMA.

Fig. 2 shows an exemplary chromatographic separation of a plasma sample from a patient.

Fig. 3 chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline,

DMA for 5% PFBOyl 10 μΙ.

Fig. 4: chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline, DMA for 10% PFBOyl 10 µΙ.

Fig. 5: chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline, DMA for 20% PFBOyl 10 µΙ.

Fig. 6: chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline, DMA for 30% PFBOyl 10 µΙ.

Fig. 7: chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline, DMA for 10 mM Na ₂ CO ₃ 10 μΙ.

Fig. 8 chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline,

DMA for 20 mM Na ₂ CO ₃ 10pl.

Fig. 9: chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline, DMA for 40 mM Na ₂ CO ₃ 10 μΙ.

Fig. 10 chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline,

DMA with 5 minutes of shaking.

Fig. 11 chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline,

DMA at 7 minutes of shaking.

Fig. 12 chromatographic separation of L-arginine, ADMA, SDMA, L-citrulline,

DMA with 10 minutes of shaking.

PL 230 479 Β1

Example

Materials

2,3,4,5,6-Pentafluorobenzoyl chloride (PFBoylCl), dimethylamine hydrochloride (DO-DMA); deuterated dimethylamine hydrochloride (D6-DMA), sodium carbonate (Na 2 CO _3), L-arginine and L-citrulline from Sigma-Aldrich (Steinheim, Germany). Labeled D7-ADMA and D7-Arg were purchased from Cambridge Isotope Laboratories. Water, acetonitrile (ACN) and formic acid were from JTBaker (Deventer, the Netherlands).

Preparation of liquid tissue samples

100 µl of e.g. plasma was transferred to microtubes (2 ml). To each sample, 10 μΙ and mixtures of internal standards (IS D6-DMA (50 μΜ); D7-ADMA (20 μΜ); D7-Arg (100 μΜ)) and 10 μΙ Na2CO ₃ (20 mM) were added and shaken for 1 min, then 500 μ ACN and 10 μΙ PFBoylCl (10% in acetonitrile) were added. The samples were shaken for 5 min at 25 ° C and 1100 rpm. It was then centrifuged for 7 min, 21,000 RCF at 4 ° C. 100 P of supernatant was transferred to chromatographic vessels containing 100 µ of water and subjected to chromatographic separation with MS / MS detection.

Preparation of solid tissue samples

100 mg of solid tissue (porcine heart muscle) was transferred to microtubes (2 ml). 200 μΙ H2O was added to each of the samples and homogenized to a uniform consistency (approx. 1 minute). Then 10 µΙ of the mixture of internal standards [IS D6-DMA (50 µΜ) was added; D7-ADMA (20 μΜ); D7-Arg (100 µΜ)] and 500 µΙ ACN, the mixture was sonicated for 2 minutes with 5 cycles and the apparatus power set to 50%. The sample after sonication was shaken for 5 min at 25 ° C and 1100 rpm. It was then centrifuged for 5 min, 21,000 RCF at 4 ° C. 550 μ of the supernatant was transferred to new microtubes, then 300 μΙ of liquid was taken from them, to which 10 μΙ of Na2CO ₃ (20 mM) and 10 μΙ of PFBoylCl (10% in acetonitrile) were added. The samples were shaken for 5 min at 25 ° C and 1100 rpm. It was then centrifuged for 7 min, 21,000 RCF at 4 ° C. 100 .mu. of supernatant was transferred to chromatographic vessels with 100 μΙ of water and subjected to chromatographic separation.

HPLC-MS / MS analysis conditions

The method was developed on an Agilent Technologies 6420 Triple Quad LC / MS tandem mass spectrometer, equipped with an electrospray ion source (ESI) connected to an Agilent Technologies 1260 Infinity liquid chromatograph. Mass Hunter Workstation Software (Agilent Technologies) was used to operate the device and analyze the results. An Acquity UPLC HSS T3 1.8 µm, 1.0 x 50 mm column supplied by Waters was used for the analysis. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B was 0.1% formic acid in methanol.

The analyzes were carried out at a constant flow of 200 μΙ / min, in the gradient of the concentrations of the mobile phase components used for the chromatographic separation of Arg, ADMA, SDMA, L-citrulline and DMA (Table 1).

Table 1

No. Time (min) Flow (μΙ / min) %AND % B 1 0 200 90.0 10.0 2 1.0 200 90.0 10.0 3 6.0 200 65.0 35.0 4 7.0 200 40.0 60.0 5 7.50 200 10.0 90.0 6 8.00 200 10.0 90.0 7 8.05 200 90.0 10.0 8 14.50 200 90.0 10.0

PL 230 479 Β1

The chromatographic separation shown in Fig. 1 was obtained for the above presented concentration gradient.

The following ions were used for the quantitative analysis of the spectrum:

Table 2. Parent ions and fragmentation ions with fragmentation energy used for chromatographic analysis Arg, D7 Arg, ADMA, D7 ADMA, SDMA, L-citrulline, DMA and D6 DMA.

No. Relationship Mother ion Ion | fragmentary Minimum time spent on studying one fragmentation reaction (dwell time) [ms] Fragmentation energy 1 D7 ADMA 404 195 100 47 2 D7 ADMA 404 167 100 79 3 D7 ADMA 404 117 100 lii 4 D7 ADMA 404 71 100 47 5 ADMA 397 195 100 43 6 ADMA 397 117 100 107 7 ADMA 397 71 100 39 8 ADMA 397 70.1 100 31 9 SDMA 397 195 100 43 10 SDMA 397 167 100 75 11 SDMA 397 ¹¹⁷ 100 103 12 SDMA 397 71 100 35 13 D7 Arg 376 194.9 100 43 14 D7 Arg 376 166.9 100 71 15 D7 Arg 376 117 100 99 16 D7 Arg 376 77.1 100 28 17 Arginine 369 194.9 100 39

PL 230 479 Β1

18 Arginine 369 167 100 63 ^! 19 Arginine 369 116.9 100 95 twenty Arginine 369 70.0 100 35 21 D6DMA 246 194.9 100 19 22 D6 DMA 246 167 100 35 23 D6 DMA 246 117 100 59 24 D6DMA 246 92.9 100 75 25 DMA 240 194.9 100 15 26 DMA 240 166.9 100 35 27 DMA 240 117 100 55 28 DMA 240 93 100 71 29 L Citrulline 370 353 100 3 thirty L- Citrulline 370 195 100 35 31 L citrulline 370 116.9 100 99 32 L- Citrulline 370 70 100 31

The following concentration values of the individual compounds were obtained for 12 tested patient sera (3 replications of each measurement were performed) are given in Table 3.

PL 230 479 Β1

Table 3. Concentration values for the 12 tested samples obtained in the chromatographic analysis.

DMA

Patient No. Concentration [μΜ] Standard deviation Percentage standard deviation 1 7.62 0.67 8.79 2 7.95 1.55 19.47 3 7.57 1.17 15.48 4 7.70 0.09 1.18 5 7.30 0.15 2.02 6 7.49 0.25 3.27 7 9.42 1.09 11.62 8 7.29 0.08 1.08 9 9.51 1.87 19.69 10 8.27 0.42 5.07 11 10.50 1.56 14.88 12 11.82 1.13 9.58 13 8.40 0.29 3.48 14 8.29 1.33 15.99 15 6.66 0.14 2.05

Mean value for 15 selected patients

8.38 [μΜ]

PL 230 479 Β1

ADMA

Patient No. Concentration [μΜ] Standard deviation Percentage standard deviation 1 0.59 0.01 1.88 2 0.44 0.01 1.34 3 0.42 0.01 2.97 4 0.47 0.03 6.43 5 0.52 0.00 0.36 6 0.44 0.06 13.77 7 0.58 0.03 4.78 8 0.46 0.02 3.45 9 0.56 0.01 2.30 10 0.43 0.02 5.27 11 0.45 0.01 1.56 12 0.47 0.01 1.99 13 0.76 0.07 8.98 14 0.55 0.07 ^r 12.11 15 0.61 0.02 3.34

Mean value for 15 selected patients

0.52 [μΜ]

PL 230 479 Β1

SDMA

Patient No. Concentration [μΜ] Standard deviation Percentage standard deviation 1 0.46 0.00 0.15 2 0.38 0.03 8.13 3 0.35 0.01 3.41 4 0.38 0.06 15.89 5 0.47 0.01, 1.57 6 0.44 0.04 8.68 7 0.64 0.08 13.05 8 0.37 0.01 3.28 9 0.66 0.00 0.43 10 0.37 0.03 7.95 11 0.28 0.00 1.60 12 0.38 0.02 5.90 13 0.90 0.10 11.20 14 0.25 0.04 14.66 15 0.36 r --------------------------— 0.02 4.46

Mean value for 15 selected patients

0.45 [μΜ]

PL 230 479 Β1

L-arginine Patient No. Concentration [μΜ] Standard deviation Percentage standard deviation 1 137.06 0.13 0.09 2 96.73 0.40 0.42 3 69.95 2.04 2.91 4 170.03 17.95 10.56 5 83.30 5.66 6.80 6 104.88 16.98 16.19 7 113.89 6.54 5.74 8 129.78 2.50 1.92 9 116.68 8.41 7.21 10 90.23 6.20 6.87 11 106.99 8.25 7.71 12 57.71 2.12 3.68 13 121.31 14.78 12.19 14 193.65 25.85 13.35 15 178.64 17.22 9.64 Mean value for 15 selected patients 118.05 [μΜ]

PL 230 479 Β1

L-citrulline Patient No. Concentration [μΜ] Standard deviation Percentage standard deviation 1 107.07 3.75 3.50 2 75.66 0.28 0.36 3 52.37 0.28 0.54 4 136.85 11.99 8.76 5 63.20 6.86 10.85 6 79.43 12.04 15.15 7 86.81 3.41 3.93 8 102.99 1.01 0.98 9 88.96 3.65 4.10 10 68.53 2.78 4.06 11 81.93 5.15 6.29 12 42.82 0.29 0.68 13 91.81 11.95 13.02 14 157.25 17.59 11.19 15 145.52 11.98 8.24 Mean value for 15 selected patients 92.08 [μΜ]

PL 230 479 Β1

Claims (15)

Patent claims 1. Method for the simultaneous determination of analytes selected from the group consisting of L-arginine (L-Arg), asymmetric dimethylarginine (ASDMA), symmetric dimethylarginine (SDMA), L-citrulline and dimethylamine (DMA) in plasma, serum, urine, human solid tissues or animal and aqueous solutions in the form of their pentafluorobenzamide derivatives, characterized in that it comprises the following steps: A. the mixture of internal standards D6-DMA, D7-ADMA, D7-Arg is added to the samples of the tested material. B. the pH of the reaction mixture is stabilized with Na2CO3 in the amount of 5-30 µl and concentration from 10 mM to 40 mM, C. Acetonitrile (AcCN) is added to the mixture of reagents, the ratio of AcCN: H2O is from 3: 1 to 9: 1. D. the analytes are derivatized through the -NH group using 2,3,4,5,6 pentafluorobenzoyl chloride added to the reaction mixture in the amount of 10 μΙ and a concentration of 5-30% with a reaction time of 5-10 minutes, E. then the pentafluorobenzamide derivatives of the analytes are separated together with the internal standard with the appropriate separation technique. 2. The method according to p. The method of claim 1, characterized in that the mixture of the internal standards in (a) is D6-DMA (1 μ 200 - 200 μΜ); D7-ADMA (4 μΜ - 100 μΜ); D7-Arg (10 μΜ - 250 μΜ). 3. The method according to p. The method of claim 2, wherein the mixture of the internal standards in (a) is D6-DMA (50 µΜ); D7-ADMA (20 μΜ); D7-Arg (100 μΜ). 4. The method according to p. The process of claim 1, wherein the pH of the reaction mixture is stabilized with 10 μΙ of Na2CO3. 5. The method according to p. The process of claim 1, wherein the concentration of AcCN is from 15-100%. 6. The method according to p. 5. The process of claim 5, wherein the concentration of AcCN is 100%. 7. The method according to p. The process of claim 1, wherein the AcCN: sample ratio is 5: 1. 8. The method according to p. The process of claim 1, wherein the concentration of 2,3,4,5,6 pentafluorobenzoyl chloride in acetonitrile is 10%. 9. The method according to p. The process of claim 1, wherein the reaction mixture formed in (d) after the addition of 2,3,4,5,6 pentafluorobenzoyl chloride is shaken for 3-10 min. 10. The method according to p. 9. The process of claim 9, wherein the reaction mixture is shaken for 5 min. 11. The method according to p. A process as claimed in claim 9 or 10, characterized in that the reaction mixture is shaken at 25 ° C. 12. The method according to p. The process of claim 9 or 10, wherein the reaction mixture is shaken at 1100 rpm. 13. The method according to p. The method of claim 1, characterized in that the separation in (d) is carried out by liquid chromatography using a detector - mass spectrometer or a tandem mass spectrometer or detectors such as LJV / VIS, DAD, FLD. 14. The method according to p. The method of claim 1, characterized in that the analysis is carried out in a reverse phase with a concentration gradient: 0.1% formic acid in water and 0.1% formic acid in methanol. 15. The method according to p. The process of claim 1, characterized in that the separation in (d) is carried out by gas chromatography after removing residual water in the sample first.