HPLC (High Performance Liquid Chromatography) efficient quantitative method for methionine and guanidinoacetic acid in whole-cell biocatalytic synthesis creatine substrate

Abstract

The invention relates to an HPLC (High Performance Liquid Chromatography) high-efficiency quantitative method for methionine and guanidinoacetic acid in a creatine substrate synthesized by whole-cell biological catalysis. According to the method, an amino bonded silica gel chromatographic column is selected, the length of the chromatographic column is 250mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of filler is 5mu m. A mobile phase A is 20 mmol of a dipotassium phosphate solution, the pH value is adjusted to 6.0-8.0 by using phosphoric acid, a mobile phase B is acetonitrile, and gradient elution is performed; the detection wavelength is 200 to 210 nm; the column temperature is 25-35 DEG C; the flow velocity is 0.7 to 1.0 mL / min. The detection method can be applied to accurate quantification of methionine and guanidinoacetic acid in a whole-cell biocatalytic synthesis creatine substrate, is simple in sample pretreatment, high in recovery rate, simple to operate, low in detection cost and short in detection time, has high-throughput sample detection capability, and is suitable for large-scale popularization and application. And a favorable tool is provided for exploring a safe and environment-friendly method for synthesizing creatine through industrial whole-cell biological catalysis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry technology, and particularly relates to a HPLC high-efficiency quantitative method for synthesizing methionine and guanidinoacetic acid in creatine substrate by whole-cell biological catalysis. BACKGROUND

[0002] Creatine is a natural amino acid derivative, which plays a key role in maintaining ATP levels in high-metabolic-activity tissues (especially muscles and brains), and is commonly used in functional foods and medicines. Creatine can accelerate the synthesis of human body protein, reduce cholesterol, blood lipids and blood sugar and delay the aging of the body, and plays a role when the energy demand is high. By supplementing creatine, the human body can increase creatine reserves and improve the level of phosphocreatine in muscles, thereby enhancing the performance of short-term, high-intensity exercise.

[0003] At present, the synthesis of creatine in industry mainly relies on chemical synthesis, which has problems such as long reaction time, complex operation steps and low product purity. In the production process, highly toxic cyanamide needs to be used, or suboptimal results are caused, which causes significant safety hazards and increases production costs. In addition, creatine synthesized by chemical methods has a bitter taste as a characteristic, which is not suitable for direct use as a food additive. Subsequently, debittering agents need to be used for treatment, which leads to a complex treatment process and a harvest rate of only 50%. Compared with traditional organic chemical production, the use of microbial production provides a safer and more environmentally friendly production method.

[0004] Whole-cell biocatalysis provides a promising approach for efficient and sustainable production of creatine. This method has many advantages, including high productivity, cost-effectiveness, simplified downstream processing, and improved product recovery. Researchers have used Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum as whole-cell biocatalysts to synthesize creatine. The typical synthesis pathway of creatine consists of two steps: the first step is the formation of guanidinoacetic acid from arginine and glycine under the catalysis of glycine amidinotransferase; the second step is the reaction of ATP with methionine to form the activated form of methionine, S-adenosyl methionine, while releasing pyrophosphate. S-adenosyl methionine catalyzes the methylation of guanidinoacetic acid under the catalysis of guanidinoacetate N-methyltransferase to form creatine (as described in "Li C, Sun P, Wei G, Zhu Y, Li J, Liu Y, Chen J, Deng Y. Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum. Synth Syst Biotechnol. 2024 Jan 9;9(1):99-107.").

[0005] The existing technology only has liquid chromatography-mass spectrometry detection method to simultaneously separate and determine methionine and guanidinoacetic acid, but the operation steps are complex, the sample processing time is long, and the reagent preparation conditions are high. There is no reported method for rapidly and simultaneously determining the concentrations of methionine and guanidinoacetic acid in the whole-cell biocatalysis synthesis of creatine catalytic liquid, which is not conducive to exploring a safe and environmentally friendly method for industrial-scale production of creatine. Therefore, there is an urgent need for an effective method for simultaneously and rapidly separating and determining the concentrations of methionine and guanidinoacetic acid in the whole-cell biocatalysis synthesis of creatine catalytic liquid. SUMMARY

[0006] To solve the above technical problems, according to the properties of methionine and guanidinoacetic acid, a chromatographic column with good retention is selected, and a gradient elution method is finally established to establish a high-performance liquid chromatography (HPLC) method for quantitatively determining methionine and guanidinoacetic acid in the whole-cell biocatalysis synthesis of creatine substrate.

[0007] To achieve the above invention purposes, the technical solution adopted by the present application is: a detection method for methionine and guanidinoacetic acid in the whole-cell biocatalysis synthesis of creatine substrate, the detection method is high-performance liquid chromatography, and the chromatographic conditions are:

[0008] Chromatographic column: amino-bonded silica gel column;

[0009] Column temperature: 25-35 °C;

[0010] Mobile phase: mobile phase A is 20 mmol potassium phosphate dibasic solution (pH adjusted to 6.0-8.0 with phosphoric acid), mobile phase B is acetonitrile, gradient elution is adopted, and the running time is 12 min;

[0011] Flow rate: 0.7-1.0 mL / min;

[0012] Detection wavelength: 200-210 nm;

[0013] Preferably, the chromatographic column uses Luna@5 μm NH2 Column temperature: 30 °C; flow rate: 0.7 mL / min; detection wavelength: 205 nm.

[0014] Preferably, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

[0015] Preferably, mobile phase A is 20 mmol potassium phosphate dibasic solution (pH adjusted to 7.0 with phosphoric acid), mobile phase B is acetonitrile, and the gradient elution conditions are 0-4 min: 50% B; 4-6 min: 49% B; 6-10.1 min: 50% B; 10.1-12 min: 50% B.

[0016] The detection method for methionine and guanidino acetic acid in the whole-cell biological catalytic synthesis of creatine substrate described above comprises the following steps:

[0017] S1. Precisely weigh guanidino acetic acid and methionine standard products, dissolve them with ultrapure water, and then dilute them with an initial mobile phase to obtain a mixed standard solution of methionine and guanidino acetic acid, wherein the initial mobile phase is a mixed solution of 20 mmol potassium phosphate dibasic solution (pH adjusted to 7.0 with phosphoric acid) and acetonitrile at a volume ratio of 1:1;

[0018] S2. Perform high-performance liquid chromatography detection on the mixed standard solution under the chromatographic conditions, and draw standard curves of methionine and guanidino acetic acid standard products according to the obtained peak areas to obtain a concentration-peak area linear regression equation of methionine and guanidino acetic acid standard products;

[0019] S3. Detect the sample to be tested under the chromatographic conditions, substitute the obtained peak area into the concentration-peak area linear regression equation, multiply by the dilution factor, and calculate the content of methionine and guanidino acetic acid in the sample to be tested.

[0020] Preferably, the mixed standard solution of methionine and guanidino acetic acid in step S1 is at least 5 portions, wherein the concentration of methionine ranges from 0.4 to 1.4 mg / mL, and the concentration of guanidino acetic acid ranges from 0.2 to 1.0 mg / mL.

[0021] Preferably, the sample to be tested in step S3 is a whole-cell biological catalysis substrate for synthesizing creatine from methionine and guanidinoacetic acid, and the preparation method of the catalysis liquid is as described in the document “Li C, Sun P, Wei G, Zhu Y, Li J, Liu Y, Chen J, Deng Y. Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum. Synth Syst Biotechnol. 2024 Jan 9; 9(1): 99-107”. Before detection, the catalysis liquid is pretreated by centrifugation at 3000 rpm for 10 min, and the supernatant is taken.

[0022] Preferably, after centrifugation of the catalysis liquid to take the supernatant, the supernatant is diluted with a solvent having the same composition as the initial mobile phase, and the initial mobile phase is a mixed solution of 20 mmol of potassium phosphate dibasic solution (pH adjusted to 7.0 with phosphoric acid) and acetonitrile at a volume ratio of 1:1.

[0023] Preferably, the sample to be tested in step S3 needs to be diluted to a methionine concentration range of 0.4-1.4 mg / mL and a guanidinoacetic acid concentration range of 0.2-1.0 mg / mL.

[0024] Preferably, in steps S2 and S3, the injection volume of the standard solution and the sample to be tested is 5 μL.

[0025] The structural formula of methionine and guanidinoacetic acid is as follows:

[0026]

[0027] The significant advantages of the present application are:

[0028] (1) A HPLC high-efficiency quantitative method for methionine and guanidinoacetic acid in a whole-cell biological catalysis substrate for synthesizing creatine, which adopts a Luna@5 μm NH2 amino-bonded silica gel chromatographic column with a column length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm, which can effectively separate methionine and guanidinoacetic acid in the catalysis liquid, and also effectively separate other related impurities.

[0029] (2) The detection technology provided by the present application has good separation degree, high accuracy, and good stability, and can better achieve the purpose of accurate quantification of methionine and guanidinoacetic acid in whole-cell biological catalysis synthesis of creatine.

[0030] (3) The detection technology provided by the application has simple pretreatment of catalytic liquid samples, high recovery rate, simple operation, low detection cost, short detection time, high detection efficiency, and high sample detection capacity. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 High performance liquid chromatogram of methionine and guanidinoacetic acid mixed standard solution in Example 1;

[0032] Figure 2 High performance liquid chromatogram of 2.3.1 methionine and guanidinoacetic acid mixed standard solution in Example 2;

[0033] Figure 3 Standard curve of methionine determined by the application in 2.3.1 of Example 2;

[0034] Figure 4 Standard curve of guanidinoacetic acid determined by the application in 2.3.1 of Example 2;

[0035] Figure 5 High performance liquid chromatogram of 2.3.2 sample to be tested in Example 2;

[0036] Figure 6 2.3.3 Precision investigation result graph in Example 2;

[0037] Figure 7 2.3.4 Stability investigation result graph in Example 2;

[0038] Figure 8 Chromatogram of blank sample (upper graph) and mixed standard (lower graph) determined by the online derivatization amino acid detection method using Agilent AdvanceBio AAA chromatographic column in Example 4;

[0039] Figure 9 Chromatogram of sample to be tested (upper graph) and mixed standard (lower graph) determined by the direct detection method using C18 chromatographic column in Example 5. DETAILED DESCRIPTION

[0040] Unless otherwise defined, all scientific and technical terms used in the application have the same meaning as commonly understood by one of ordinary skill in the art to which the application relates.

[0041] The disclosures of various publications, patents and published patent specifications referred to herein are hereby incorporated by reference in their entireties.

[0042] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of the present application.

[0043] Embodiment one: simultaneous detection and analysis of methionine and guanidinoacetic acid mixed standard using the method of the present application

[0044] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph

[0045] Methionine standard (chromatographic grade 99.9%), guanidinoacetic acid standard (chromatographic grade 98.7%), dipotassium hydrogen phosphate (analytical pure), phosphoric acid (analytical pure), acetonitrile (chromatographic pure), ultrapure water.

[0046] 1.2 Chromatographic conditions

[0047] Chromatographic column: Luna@5 μm NH2 The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm;

[0048] Chromatographic column temperature: 30℃;

[0049] Mobile phase: mobile phase A is 20 mmol dipotassium hydrogen phosphate solution, adjusted to pH 7.0 with phosphoric acid, and mobile phase B is acetonitrile, using gradient elution, with a running time of 12 min.

[0050] The gradient elution conditions are 0-4 min: 50% B; 4-6 min: 49% B; 6-10.1 min: 50% B; 10.1-12 min: 50% B.

[0051] Flow rate: 0.7 mL / min;

[0052] Injection volume: 5 μL;

[0053] Detection wavelength: 205 nm.

[0054] 1.3 Operation steps

[0055] Accurately weigh 10.11 mg of methionine standard and 10.67 mg of guanidinoacetic acid standard into a 10 mL volumetric flask, dissolve with ultrapure water, and then dilute to the initial mobile phase volume with the same solvent as the initial mobile phase, which is a 20 mmol potassium phosphate dibasic solution with a pH adjusted to 7.0 with phosphoric acid, mixed with acetonitrile at a volume ratio of 1:1, and shaken well to obtain a mixed standard solution with a methionine concentration of 1.0103 mg / mL and a guanidinoacetic acid concentration of 1.0531 mg / mL.

[0056] Take the mixed standard solution, microfilter it into a sample bottle with a 0.22 μm organic filter membrane, and perform high-performance liquid chromatography analysis according to the chromatographic conditions described in 1.2, and record the chromatogram.

[0057] The results are shown in the accompanying Figure 1 , where 5.287 min is methionine and 6.528 min is guanidinoacetic acid. It can be seen that under these conditions, the methionine peak can be completely separated from the guanidinoacetic acid peak, and the peak shape has good symmetry.

[0058] Example 2: Simultaneous detection of catalytic liquid for whole-cell biological catalytic synthesis of creatine using the method of the present application, and preparation of a standard curve

[0059] 2.1 Instruments and reagents

[0060] High-performance liquid chromatograph: Agilent 1260 Infinity II high-performance liquid chromatograph;

[0061] Methionine standard (chromatographic grade 99.9%), guanidinoacetic acid standard (chromatographic grade 98.7. %), potassium phosphate dibasic (analytical pure), phosphoric acid (analytical pure), acetonitrile (chromatographic pure), ultrapure water.

[0062] 2.2 Chromatographic conditions

[0063] Chromatographic column: Luna@5 μm NH2 The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm;

[0064] Chromatographic column temperature: 30°C;

[0065] Mobile phase: Mobile phase A is a 20 mmol potassium phosphate dibasic solution (pH adjusted to 7.0 with phosphoric acid), and mobile phase B is acetonitrile, using gradient elution with a run time of 12 min.

[0066] The gradient elution conditions are 0-4 min: 50% B; 4-6 min: 49% B; 6-10.1 min: 50% B; 10.1-12 min: 50% B.

[0067] Flow rate: 0.7 mL / min;

[0068] Injection volume: 5 μL;

[0069] Detection wavelength: 205 nm.

[0070] 2.3 Operation steps

[0071] 2.3.1 Draw standard curve

[0072] Accurately weigh 20.41 mg of methionine standard and 10.67 mg of guanidinoacetic acid standard into a 10 mL volumetric flask, dissolve with ultrapure water, and then dilute to the initial mobile phase, which is a 20 mmol potassium phosphate dibasic solution, adjusted to pH 7.0 with phosphoric acid, mixed with acetonitrile at a volume ratio of 1:1. Shake well to obtain a mixed standard solution mother liquor with a methionine concentration of 2.0206 mg / mL and a guanidinoacetic acid concentration of 1.0531 mg / mL.

[0073] Take the mixed standard solution mother liquor described above and dilute it with the initial mobile phase to obtain five concentration points for each of methionine and guanidinoacetic acid, with concentrations of 0.4041 mg / ml, 0.8082 mg / ml, 1.0103 mg / ml, 1.2123 mg / ml, 1.4144 mg / ml for methionine and 0.2106 mg / ml, 0.4212 mg / ml, 0.6320 mg / ml, 0.8425 mg / ml, 1.0531 mg / ml for guanidinoacetic acid.

[0074] Take the five concentration point gradient standard solutions of methionine and guanidinoacetic acid described above, and microfilter them into sample bottles using a 0.22 μm organic filter membrane. Perform high performance liquid chromatography analysis under the chromatographic conditions described in 2.2.

[0075] The results of the mixed standard solution concentration and peak area are shown in Tables 1 and 2. The theoretical plate number of each chromatogram is greater than 5000, and the separation degree from the impurity peaks is good. The chromatogram of the mixed standard solution with a methionine concentration of 1.2123 mg / mL and a guanidinoacetic acid concentration of 0.8425 mg / mL is shown in Figure 1. Figure 2

[0076] Table 1 Methionine standard curve test results

[0077] Concentration (mg / mL) Peak area (mAU-s) 0.4041 2433.293 0.8082 4808.904 1.0103 5900.042 1.2123 7144.468 1.4144 8202.146

[0078] Table 2 Guanidinoacetic acid standard curve test results

[0079]

[0080]

[0081] ​The standard curve of methionine and guanidinoacetic acid standard was drawn according to the standard concentration and the corresponding peak area, with the concentration as the abscissa and the peak area as the ordinate, and the concentration-peak area linear regression equation of the methionine standard was y = 5812.1380x + 50.6753, R 2 = 0.99968; and the concentration-peak area linear regression equation of the guanidinoacetic acid standard was y = 7078.5887x + 72.0659, R 2 = 0.99963. Among them, Figure 3 is the methionine standard curve, Figure 4 is the guanidinoacetic acid standard curve.

[0082] From the results of this embodiment, it is shown that by determining the methionine and guanidinoacetic acid standard solution, the method of the present application has a good linear relationship (R 2 = 0.99968) for detecting methionine in the range of 0.4-1.4 mg / mL, and also has a good linear relationship (R 2 = 0.99963) for detecting guanidinoacetic acid in the range of 0.2-1.0 mg / mL.

[0083] 2.3.2 Detection of the sample to be tested

[0084] Take 0.5 ml of whole-cell biological catalysis substrate, which is the catalytic liquid for synthesizing creatine from methionine and guanidinoacetic acid (the preparation method is described in Li C, Sun P, Wei G, Zhu Y, Li J, Liu Y, Chen J, Deng Y. Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum. Synth Syst Biotechnol. 2024 Jan 9; 9(1): 99-107), centrifuge at 3000 rpm for 10 min, take the supernatant, dilute to 5 ml with the same solvent as the initial mobile phase, the initial mobile phase is 20 mmol potassium phosphate buffer solution, adjust the pH to 7.0 with phosphoric acid, mix with acetonitrile at a volume ratio of 1:1, microfiltration to the sample bottle with a 0.22 μm organic filter membrane, and obtain the sample to be tested.

[0085] Under the same detection conditions as the standard solution, the sample to be tested is detected, and the chromatogram is as shown in Figure 5As shown, the peak area of ​​methionine was 6018.313. Substituting this peak area into the above linear regression equation of methionine concentration-peak area and multiplying by the dilution factor of 10, the content of methionine in the sample was calculated to be 10.27 mg / mL. The peak area of ​​guanidinoacetic acid was 4228.941. Substituting this peak area into the above linear regression equation of guanidinoacetic acid concentration-peak area and multiplying by the dilution factor of 10, the content of guanidinoacetic acid in the sample was calculated to be 5.87 mg / mL.

[0086] 2.3.3 Precision Examination

[0087] Take the above-mentioned mixed standard solution of methionine (1.0103 mg / mL) and guanidinoacetic acid (0.6320 mg / mL) and perform high-performance liquid chromatography (HPLC) analysis under the above chromatographic conditions. Repeat the analysis 6 times. See [link to HPLC analysis]. Figure 6 The peak areas of methionine obtained were 5141.602, 5145.107, 5126.734, 5114.620, 5096.777, and 5122.441, with an average value of 5124.547 and an RSD of 0.348%. The peak areas of guanidinoacetic acid obtained were 4062.985, 4215.477, 4162.079, 4135.884, 4125.631, and 4153.759, with an average value of 4142.635 and an RSD of 1.21%. This indicates that the high-performance liquid chromatography (HPLC) method for the detection of methionine and guanidinoacetic acid provided in this embodiment has good precision.

[0088] 2.3.4 Stability Assessment

[0089] 0.5 ml of a catalytic solution for the synthesis of creatine from methionine and guanidinoacetic acid, a whole-cell biocatalytic substrate, was centrifuged at 3000 rpm for 10 min. The supernatant was collected and diluted to 5 ml with a solvent of the same composition as the initial mobile phase (20 mmol dipotassium hydrogen phosphate solution, pH adjusted to 7.0 with phosphoric acid, and mixed with acetonitrile at a 1:1 volume ratio). The solution was then microfiltered through a 0.22 μm organic filter into a sample vial to obtain the test sample. High-performance liquid chromatography (HPLC) analysis was performed at 0 h, 4 h, 8 h, and 12 h after preparation, according to the above chromatographic conditions. Stability results are shown in the figure. Figure 7The peak area of methionine was 6018.313, 5980.492, 5969.925, 5956.885, the average value was 5981.403, and the RSD was 0.442%, the retention time was 5.291 min, 5.286 min, 5.291 min, 5.291 min, the average value was 5.290 min, and the RSD was 0.05%; the peak area of guanidine acetic acid was 4228.941, 4221.691, 4215.914, 4196.905, the average value was 4215.863, and the RSD was 0.325%, the retention time was 6.537 min, 6.536 min, 6.545 min, 6.548 min, the average value was 6.541 min, and the RSD was 0.09%, indicating that the high performance liquid chromatography detection method of methionine and guanidine acetic acid provided in the embodiment has good stability.

[0090] 2.3.5 Limit of quantification

[0091] Precisely take 10.11 mg of methionine standard into a 10 mL volumetric flask, dissolve with ultrapure water, and then dilute with the initial mobile phase. High performance liquid chromatography analysis was carried out according to the above chromatographic conditions, and the peak height was 849.352 and the baseline noise was 0.19. The methionine standard solution was diluted 400 times, and detection was carried out under the same detection conditions as the standard solution, and the peak height was 0.191. Therefore, the limit of quantification of this method was 0.002527 mg / mL (calculation formula: 10.11 mg / 10 mL / 400). The high performance liquid chromatography detection method of methionine provided in the embodiment has a low detection limit.

[0092] Precisely take 10.67 mg of guanidine acetic acid standard into a 10 mL volumetric flask, dissolve with ultrapure water, and then dilute with the initial mobile phase. High performance liquid chromatography analysis was carried out according to the above chromatographic conditions, and the peak height was 885.101 and the baseline noise was 0.35. The guanidine acetic acid standard solution was diluted 200 times, and detection was carried out under the same detection conditions as the standard solution, and the peak height was 3.731. Therefore, the limit of quantification of this method was 0.005335 mg / mL (calculation formula: 10.67 mg / 10 mL / 200). The high performance liquid chromatography detection method of guanidine acetic acid provided in the embodiment has a low detection limit.

[0093] Example Three: Recovery rate determination

[0094] Take the whole cell biological catalysis substrate for the synthesis of creatine from methionine and guanidino acetic acid 10 ml, centrifuge at 3000 rpm for 10 min, take the supernatant, dilute to 200 ml with the same solvent as the initial mobile phase, the initial mobile phase is 20 mmol potassium phosphate solution, adjust the pH to 7.0 with phosphoric acid, mix with acetonitrile at a volume ratio of 1:1 to form, take out 9 parts, each 10 mL; respectively take 2 mg, 3 mg, 6 mg of methionine standard each 3 parts, add to the above 9 parts of sample solution respectively, dissolve and mix, filter to the sample bottle with 0.22 μm organic filter membrane, carry out high performance liquid chromatography analysis under the same chromatographic conditions as in examples 1 and 2, detect the content of methionine standard in each solution, calculate the recovery rate, the results are shown in table 3.

[0095] Similarly, take the whole cell biological catalysis substrate for the synthesis of creatine from methionine and guanidino acetic acid 10 ml, centrifuge at 3000 rpm for 10 min, take the supernatant, dilute to 200 ml with the same solvent as the initial mobile phase, the initial mobile phase is 20 mmol potassium phosphate solution, adjust the pH to 7.0 with phosphoric acid, mix with acetonitrile at a volume ratio of 1:1 to form, take out 9 parts, each 10 mL; respectively take 2 mg, 3 mg, 6 mg of methionine standard each 3 parts, add to the above 9 parts of sample solution respectively, dissolve and mix, filter to the sample bottle with 0.22 μm organic filter membrane, carry out high performance liquid chromatography analysis under the same chromatographic conditions as in examples 1 and 2, detect the content of methionine standard in each solution, calculate the recovery rate, the results are shown in table 3.

[0096] Table 3 recovery rate and relative standard deviation of methionine

[0097]

[0098] Table 4 recovery rate and relative standard deviation of guanidino acetic acid

[0099]

[0100]

[0101] From table 3 and table 4, in 9 recovery tests, the average recovery rate of methionine is 99.51%, RSD = 0.38% (n = 9); the average recovery rate of guanidino acetic acid is 98.62%, RSD = 0.70% (n = 9), the recovery rate is greater than 98%, which indicates that the high performance liquid detection method of methionine and guanidino acetic acid provided in this embodiment has good recovery rate.

[0102] Example four: using Agilent AdvanceBio AAA chromatographic column to detect methionine and guanidino acetic acid by online derivatization of amino acid

[0103] Automatic online pre-column amino acid derivatization and chromatographic separation for simultaneous determination of methionine and guanidino acetic acid were performed by Agilent autosampler and Agilent AdvanceBio AAA column packed with surface porous particles. Primary amino acids were derivatized with o-phthaldialdehyde (OPA), which is a commonly used method for primary amino acid detection, and multiple amino acids can be separated and determined simultaneously.

[0104] 4.1 Instruments and reagents

[0105] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph;

[0106] Methionine standard (chromatographic grade 99.9%), guanidino acetic acid standard (chromatographic grade 98.7. %), disodium hydrogen phosphate (analytical pure), sodium borate (analytical pure), hydrochloric acid (analytical pure), acetonitrile (chromatographic pure), methanol (chromatographic pure), AdvanceBio amino acid analysis kit (5190-9426), ultrapure water.

[0107] 4.2 Chromatographic conditions

[0108] Chromatographic column: Agilent AdvanceBio AAA C18, with a column length of 100 mm, an inner diameter of 4.6 mm, and a packing particle size of 2.7 μm;

[0109] Chromatographic column temperature: 40℃;

[0110] Mobile phase: A) 10 mmol / L disodium hydrogen phosphate and 10 mM sodium borate solution, pH adjusted to 8.2 with hydrochloric acid, B) methanol: acetonitrile: water, 45:45:10 (v:v:v), gradient elution, run time 12 min.

[0111] The gradient elution conditions were as follows: 0-8 min: 10-57% B; 8-8.5 min: 57-100% B; 8.5-9.5 min: 100% B; 9.5-9.6 min: 100-15% B; 9.6-11 min: 15% B; 11-12 min: 15-10% B.

[0112] Flow rate: 1.5 mL / min;

[0113] Injection volume: 20 μL;

[0114] Detection wavelength: 338 nm.

[0115] Automatic sampler program settings:

[0116] Take 2.5 μL of borate buffer

[0117] · Take 1.0 μL of sample

[0118] • Mix 3.5 μL of the mixture 5 times in the wash port

[0119] • Wait 0.2 minutes, then aspirate 0.5 μL of OPA

[0120] • Mix 4 μL of the mixture 10 times in the wash port

[0121] • Aspirate 0.4 μL of FMOC

[0122] • Mix 4.4 μL of the mixture 10 times in the wash port

[0123] • Aspirate 32 μL of diluent

[0124] • Mix 20 μL of the mixture 8 times in the wash port

[0125] • Inject

[0126] • Wait 0.1 minutes

[0127] • Switch valve to bypass.

[0128] 4.3 Procedure

[0129] Accurately weigh 10.11 mg of methionine standard and 10.67 mg of guanidinoacetic acid standard into a 100 mL volumetric flask. Dissolve with ultrapure water and make up to volume with the same solvent as the initial mobile phase, which is A) 10 mmol / L disodium hydrogen phosphate and 10 mM sodium borate solution, pH adjusted to 8.2 with hydrochloric acid and B) methanol:acetonitrile:water = 45:45:10 (v:v:v) mixed at a volume ratio of 9:1, and shake well to obtain a mixed standard solution with a methionine concentration of 0.1010 mg / mL and a guanidinoacetic acid concentration of 0.1053 mg / mL. Take the mixed standard solution and microfilter it into a sample injection bottle with a 0.22 μm organic filter membrane.

[0130] Take 0.5 mL of the catalytic liquid of the whole-cell biological catalysis substrate, which is the synthesis of creatine from methionine and guanidinoacetic acid, centrifuge at 3000 rpm for 10 min, take the supernatant, dilute to 5 mL with the same solvent as the initial mobile phase, which is A) 10 mmol / L disodium hydrogen phosphate and 10 mM sodium borate solution, pH adjusted to 8.2 with hydrochloric acid and B) methanol:acetonitrile:water = 45:45:10 (v:v:v) mixed at a volume ratio of 9:1, and microfilter it into a sample injection bottle with a 0.22 μm organic filter membrane to obtain the sample to be tested.

[0131] The initial mobile phase was microfiltered into the sample vial using a 0.22 μm organic filter membrane as a blank control. The initial mobile phase consisted of A) a 10 mmol / L disodium hydrogen phosphate and 10 mM sodium borate solution, with the pH adjusted to 8.2 using hydrochloric acid, and B) a mixture of methanol, acetonitrile, and water in a volume ratio of 45:45:10 (v:v:v) at a ratio of 9:1.

[0132] The mixed standard solution, the test sample, and the blank solvent were detected using the chromatographic conditions and pre-column derivatization method described in 4.2, and the chromatograms were recorded.

[0133] The results of the representative mixed standard and blank control are attached. Figure 8 The peak at 6.076 min in the figure represents methionine, and the peak at 11.165 min represents the blank solvent peak. Guanidinylacetic acid was not detected in either the mixed standard or the test sample. These results indicate that this amino acid derivatization method can only detect methionine; guanidinylacetic acid cannot be derivatized with o-phthalaldehyde (OPA), therefore, simultaneous quantitative analysis of guanidinylacetic acid and methionine is not possible.

[0134] Example 5: Direct detection of methionine and guanidinoacetic acid using a C18 column

[0135] The chromatographic column used was an Agilent Ecipse XDB-C18 with a length of 150 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm. Other chromatographic conditions were the same as in Example 1.

[0136] 5.1 Operating Procedures

[0137] Accurately weigh 10.11 mg of methionine standard and 10.67 mg of guanidinoacetic acid standard into a 10 mL volumetric flask, dissolve them in ultrapure water, and then dilute to volume with the same solvent as the initial mobile phase, wherein the initial mobile phase is 20 mmol dipotassium hydrogen phosphate solution, the pH is adjusted to 7.0 with phosphoric acid, and mixed with acetonitrile at a volume ratio of 1:1. Shake well to obtain a mixed standard solution with a methionine concentration of 1.0103 mg / mL and a guanidinoacetic acid concentration of 1.0531 mg / mL. Microfilter the solution into a sample vial using a 0.22 μm organic filter membrane.

[0138] Take 0.5 ml of the catalytic solution for the synthesis of creatine from methionine and guanidinoacetic acid, a whole-cell biocatalytic substrate, centrifuge at 3000 rpm for 10 min, collect the supernatant, and dilute to 5 ml with a solvent of the same composition as the initial mobile phase. The initial mobile phase is a 20 mmol dipotassium hydrogen phosphate solution, adjusted to pH 7.0 with phosphoric acid, and mixed with acetonitrile at a 1:1 volume ratio. Microfilter the solution through a 0.22 μm organic filter membrane into a sample vial to obtain the sample to be tested.

[0139] Take the mixed standard solution and the sample to be tested, and perform high performance liquid chromatography analysis according to the above chromatographic conditions, and record the chromatogram.

[0140] The representative mixed standard and sample to be tested are detected, and the results are shown in the following table. Figure 9 Cysteine and guanidino acetic acid are not detected in the mixed standard and the sample to be tested. The results show that the chromatographic column does not retain cysteine and guanidino acetic acid, and thus cannot determine guanidino acetic acid and cysteine.

[0141] It can be seen from the comparison of examples 1, 2 and examples 4, 5 that the conventional amino acid derivatization method and the ordinary C18 chromatographic column cannot simultaneously detect guanidino acetic acid and cysteine, while the amino-bonded silica gel chromatographic column and the chromatographic method provided by the present application can simultaneously detect guanidino acetic acid and cysteine, and can accurately quantify the two.

[0142] The above only describes the preferred embodiments of the present application, and does not limit the present application. Any modification, equivalent replacement, etc. within the spirit and principles of the present application should be included in the protection scope of the present application.

[0143] The foregoing examples and methods described in the present application can be different based on the ability, experience and preference of the person skilled in the art.

[0144] The steps of the method in the present application are only listed in a certain order, which does not constitute any limitation on the order of the steps of the method.

Claims

1. A method for detecting the catalytic synthesis of methionine and guanidinoacetic acid from creatine substrates by whole-cell organisms, characterized in that, The detection method is high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography are as follows: The chromatographic column is an amino-bonded silica gel column. The column temperature of the chromatographic column is 25-35°C. The mobile phase A is 20 mmol of dipotassium hydrogen phosphate solution, and the pH is adjusted to 6.0-8.0 by phosphoric acid; the mobile phase B is acetonitrile, and gradient elution is adopted, and the running time is 12 min. The flow rate is 0.7-1.0 mL / min. The detection wavelength is 200-210 nm.

2. The method of claim 1, wherein: Chromatographic column Luna® 5 μm NH2 Chromatographic column Column temperature: 30 °C; flow rate: 0.7 mL / min; detection wavelength 205 nm.

3. The method of claim 1, wherein, The length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the particle size of the packing material is 5 μm.

4. The method of claim 1, wherein, The mobile phase A is 20 mmol of dipotassium hydrogen phosphate solution, and the pH is adjusted to 7.0 by phosphoric acid; the mobile phase B is acetonitrile, and the gradient elution conditions are as follows: 0-4 min: 50% B; 4-6 min: 49% B; 6-10.1 min: 50% B; 10.1-12 min: 50% B.

5. The method according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: S1. Precisely weigh the guanidino acetic acid and methionine standard, dissolve it with ultrapure water, and then dilute it with the initial mobile phase to obtain a methionine and guanidino acetic acid mixed standard solution, wherein the initial mobile phase is a mixed solution of 20 mmol of dipotassium hydrogen phosphate solution and acetonitrile in a volume ratio of 1:1, and the pH of the dipotassium hydrogen phosphate solution is adjusted to 7.0 by phosphoric acid; S2. Perform high performance liquid chromatography detection on the mixed standard solution under the chromatographic conditions, and draw a standard curve of methionine and guanidino acetic acid standard according to the obtained peak area to obtain a concentration-peak area linear regression equation of the methionine and guanidino acetic acid standard; S3. Detect the sample to be tested under the chromatographic conditions, substitute the obtained peak area into the concentration-peak area linear regression equation, multiply by the dilution multiple, and calculate the content of methionine and guanidino acetic acid in the sample to be tested.

6. The method of claim 5, wherein, The methionine and guanidino acetic acid mixed standard solution in step S1 is at least 5 portions, wherein the concentration of methionine ranges from 0.4 to 1.4 mg / mL, and the concentration of guanidino acetic acid ranges from 0.2 to 1.0 mg / mL.

7. The method of claim 5, wherein, The sample to be tested in step S3 is a whole-cell biological catalysis substrate for synthesizing creatine from methionine and guanidino acetic acid, which is diluted with the initial mobile phase before detection.

8. The method of claim 5, wherein, The sample to be tested in step S3 is diluted to a methionine concentration ranging from 0.4 to 1.4 mg / mL and a guanidino acetic acid concentration ranging from 0.2 to 1.0 mg / mL.

9. The method of claim 5, wherein, In steps S2 and S3, the injection volume of the standard solution and the sample to be tested is 5 μL.

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