Amino acid derivatization reagent pair based on non-stable isotope labeling and preparation method and application thereof

Abstract

The invention discloses a non-stable isotope-labeled amino acid derivatization reagent pair and its preparation method and application, wherein the amino acid derivatization reagent pair is synthesized by a two-step method, wherein L-phenylalanine and paraacetaldehyde or paraformaldehyde are first refluxed to synthesize N, N-diethylphenylalanine and N, N-dimethylphenylalanine, and then the product of the first step is reacted with N-hydroxysuccinimide to synthesize N, N-dimethylphenylalanine succinimide ester and N, N-diethylphenylalanine succinimide ester. The synthesis step of the present invention is simple, low cost, no stable isotope internal standard is required, and the detection cost of chiral amino acids can be greatly reduced; in addition, the present invention can also achieve D- / L-split and internal standard quantification of more than 40 kinds of amino acids, with high sensitivity, good stability and other advantages, and can be used for splitting and detecting D- / L-amino acids in complex samples.

Description

Technical Field

[0001] The present invention relates to the field of amino acid detection, and in particular to an amino acid derivatization reagent pair based on non-stable isotope labeling, and a preparation method and application thereof. Background Art

[0002] Abnormal levels of chiral amino acids are associated with many diseases, and the presence of D-amino acids in serum or urine samples can often aid in the diagnosis of some diseases. However, the abundance of D-amino acids in biological samples is lower than that of L-amino acids, so D-amino acid quantification methods require high sensitivity and selectivity.

[0003] Chemical derivatization can achieve effective separation of D / L-amino acids on chromatographic columns, and can also achieve enhanced sensitivity of D-amino acid signals and low background signals to achieve trace detection of D-amino acids. Therefore, the combination of chemical derivatization and chromatography is currently a more common D / L-amino acid detection technology. Among them, isotope labeling has become the first choice for quantitative detection of D / L-amino acids due to its good analytical performance, but it still has the following problems: (1) There is isotope interference and mutual ionization suppression between the analyte and the internal standard, and the number of objects that can be separated is small; (2) The synthesis process of existing amino acid derivatization reagents is complicated and the cost is high.

[0004] In summary, the development of new chiral amino acid derivatization reagents is of great significance for reducing detection costs, improving the ultrasensitive signal of D / L-amino acids in mass spectrometry and rapid quantitative detection. Summary of the Invention

[0005] In view of this, the present invention proposes a non-stable isotope-labeled amino acid derivatization reagent pair, and also provides a preparation method and application of the non-stable isotope-labeled amino acid derivatization reagent pair.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The amino acid derivatization reagents based on non-stable isotope labeling described in the present invention include N,N-dimethylphenylalanine succinimide ester and N,N-diethylphenylalanine succinimide ester, and their structural formulas are:

[0008] .

[0009] The present invention also provides a method for preparing a non-stable isotope-labeled amino acid derivatization reagent pair, the preparation method comprising the following steps:

[0010] L-phenylalanine and paraacetaldehyde or paraformaldehyde are mixed together, TFE and NaBH4 are added, and the mixture is stirred under reflux conditions. After the reaction is completed, N,N-diethylphenylalanine or N,N-dimethylphenylalanine is obtained by recrystallization using methanol-acetone.

[0011] In N,N-dimethylformamide as a solvent, N,N-diethylphenylalanine or N,N-dimethylphenylalanine is reacted with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at room temperature with stirring; after the reaction is clear, N,N-diethylphenylalanine succinimide ester or N,N-dimethylphenylalanine succinimide ester is purified to obtain a white powder.

[0012] The beneficial effect is that the present invention adopts a two-step synthesis method to prepare N,N-diethylphenylalanine succinimide ester and N,N-dimethylphenylalanine succinimide ester, the synthesis is simple, the cost is low, and it is conducive to quantitative production.

[0013] The present invention also provides the use of an amino acid derivatization reagent in the determination of amino acids, wherein the use uses an amino acid derivatized with N,N-dimethylphenylalanine succinimide ester as an internal standard. Specifically, the method includes:

[0014] In the first step, N,N-diethylphenylalanine succinimidyl ester was added to the sample for derivatization reaction;

[0015] In the second step, LC-MRM is used to perform qualitative separation and quantitative detection on the derivatized test solution. The quantitative detection includes the following steps: derivatizing amino acid standards with N,N-diethylphenylalanine succinimidyl ester and N,N-dimethylphenylalanine succinimidyl ester, respectively, and mixing the derivatized amino acids at a certain volume ratio to obtain mixed derivatized amino acids with different concentration ratios;

[0016] LC-MRM was used to detect and analyze mixed derivatized amino acids at different concentration ratios. A standard curve was drawn with the concentration ratio of N,N-diethylphenylalanine succinimide ester-amino acid and N,N-dimethylphenylalanine succinimide ester-amino acid as the abscissa and the area ratio of N,N-diethylphenylalanine succinimide ester-amino acid and N,N-dimethylphenylalanine succinimide ester-amino acid as the ordinate.

[0017] A known concentration of N,N-dimethylphenylalanine succinimidyl ester-amino acid was added to the derivatized sample as an internal standard solution, and the sample was detected and analyzed using LC-MRM. The amino acid mass spectrometry parameters were used for qualitative analysis, and the amino acid content in the sample was determined based on the standard curve and the internal standard.

[0018] The liquid chromatography conditions for LC-MRM were as follows: mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, and the elution conditions were: 10%-90% B from 0 to 23 min. The mass spectrometry conditions were as follows: ESI source was in positive ion mode, multiple reaction monitoring scan mode was adopted, ISVF was set to 5500 V, GS1 was set to 55 psi, GS2 was set to 55 psi, CUR was set to 25 psi, and TEM was set to 500°C.

[0019] Preferably, the amino acids in the internal standard solution include D / L-Ala, D / L-Val, D / L-Leu, D / L-Ile, D / L-Met, D / L-Pro, D / L-Trp, D / L-Ser, D / L-Tyr, D / L-Phe, D / L-Asn, D / L-Gln, D / L-Thr, D / L-Asp, D / L-Glu, D / L-Lys, D / L-Arg, D / L-His, D / L-Homoser, D / L-Cit, D / L-Kynu, D / L-Abu and Gly.

[0020] The beneficial effects are: the derivatization reagent pair synthesized by the present invention has simple synthesis steps and low cost, and can reduce the detection cost of chiral amino acids to a certain extent; in addition, the reagent pair of the present invention can achieve the separation of 43 amino acids (including 20 pairs of D / L-amino acids) and can achieve the quantification of 51 amino acids, has high sensitivity, accuracy, precision and stability, and can be used for the separation and detection of D / L-amino acids in complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the separation chromatogram of 51 amino acids according to the present invention. DETAILED DESCRIPTION

[0022] The following describes the embodiments of the present invention in detail with reference to the accompanying drawings. It should be noted that the chemical reagents used in the present invention are commercially available. LC-MRM is employed for amino acid detection, using an LC-30A (Shimadzu, Japan) ultra-high performance liquid chromatography system and a C18 column (1.7 μm particle size, 150 × 2.1 mm). The mass spectrometer is a 6500 Q-TRAP mass spectrometer (AB SCIEX, USA) equipped with an ion-driven turbo ESI source.

[0023] The amino acids in the embodiments of the present invention include alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), phenylalanine (Phe), asparagine (Asn), glutamine (Gln), threonine (Thr), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), homoserine (Homoser), ornithine (Orn), citrulline (Cit), cis-4-hydroxy-proline (cis-4-OH-Pro), trans-4-hydroxy-proline (trans-4-OH-Pro), kynurenine (Kynu), 2-aminobutyric acid (Abu), and glycine (Gly).

[0024] Example 1 Synthesis of N,N-diethylphenylalanine succinimide ester of the present invention

[0025] Step 1: Synthesis of N,N-diethylphenylalanine

[0026] Weigh 3.3038 g of L-phenylalanine and 4.0805 g of paraacetaldehyde into a flask, then add 100 mL of TFE and 3.0264 g of NaBH4. Stir and react under reflux. After the reaction, recrystallize from methanol-acetone and dry to obtain white powder N,N-diethylphenylalanine, whose structural formula is as follows:

[0027] ;

[0028] Step 2: Synthesis of N,N-diethylphenylalanine succinimide ester

[0029] To a 25 mL flask, add 116 mg of N,N-diethylphenylalanine, 74.2 mg of N-hydroxysuccinimide, 113.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4 mL of N,N-dimethylformamide. Stir and react at room temperature for 36 h. After the reaction is clear, purify by column chromatography to obtain white powder N,N-diethylphenylalanine succinimide ester, the structural formula of which is as follows:

[0030] .

[0031] Example 2 Synthesis of N,N-dimethylphenylalanine succinimide ester according to the present invention

[0032] In the first step, the difference between this embodiment and embodiment 1 is that this embodiment uses paraformaldehyde instead of the paraacetaldehyde in embodiment 1 to synthesize N,N-dimethylphenylalanine, the structural formula of which is as follows:

[0033] ;

[0034] In the second step, the difference between this embodiment and embodiment 1 is that this embodiment uses N,N-dimethylphenylalanine instead of N,N-diethylphenylalanine in embodiment 1 to synthesize white powdery N,N-dimethylphenylalanine succinimide ester, the structural formula of which is as follows:

[0035] .

[0036] Example 3 Application of Example 1 and Example 2 in the separation and quantification of chiral amino acids

[0037] The present invention constructs a method for detecting amino acids based on the N,N-diethylphenylalanine succinimide ester in Example 1 and the N,N-dimethylphenylalanine succinimide ester in Example 2. The method uses amino acids labeled with N,N-dimethylphenylalanine succinimide ester as internal standards for quantitative detection.

[0038] The detection method of the present invention includes the following specific contents:

[0039] The first step is to draw the standard curve

[0040] S11, prepare N,N-dimethylphenylalanine succinimidyl ester solution and N,N-diethylphenylalanine succinimidyl ester solution with 500 μL of acetonitrile / water (v:v = 5:1) to a concentration of 10,000 nmol / mL;

[0041] Add D / L amino acid standard to the N,N-dimethylphenylalanine succinimide ester solution for derivatization reaction. After the reaction is completed, dilute to obtain a 400 pmol / mL N,N-dimethylphenylalanine succinimide ester-amino acid standard solution, which is used as the internal standard solution.

[0042] Add D / L amino acid standards to the N,N-diethylphenylalanine succinimide ester solution to prepare N,N-diethylphenylalanine succinimide ester-amino acid standard solutions with concentrations of 4, 10, 40, 100, 400, 1000, 4000, 10000, and 20000 pmol / mL, respectively;

[0043] Among them, the D / L amino acid standards in this example include D / L-Ala, D / L-Val, D / L-Leu, D / L-Ile, D / L-Met, D / L-Pro, D / L-Trp, D / L-Ser, D / L-Tyr, D / L-Phe, D / L-Asn, D / L-Gln, D / L-Thr, D / L-Asp, D / L-Glu, D / L-Lys, D / L-Arg, D / L-His, D / L-Homoser, D / L-Cit, D / L-Kynu, D / L-Abu and Gly, a total of 51 amino acids;

[0044] S12, mixing different volumes of a standard solution of N,N-diethylphenylalanine succinimidyl ester-amino acid and an internal standard solution to obtain mixtures with concentration ratios of 0.01:1, 0.04:1, 0.1:1, 0.25:1, 1:1, 2.5:1, 10:1, 20:1, and 50:1, and analyzing the mixtures by LC-MRM;

[0045] A standard curve was drawn with the concentration ratio of N,N-diethylphenylalanine succinimide ester-amino acid and N,N-dimethylphenylalanine succinimide ester-amino acid as the abscissa and the area ratio of N,N-diethylphenylalanine succinimide ester-amino acid and N,N-dimethylphenylalanine succinimide ester-amino acid as the ordinate;

[0046] The LC-MRM liquid chromatography conditions were as follows: mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, and the elution conditions were: 10%-90% B over 0-23 min. The mass spectrometry conditions were: ESI source in positive ion mode, multiple reaction monitoring scan mode, ISVF set to 5500 V, GS1 set to 55 psi, GS2 set to 55 psi, CUR set to 25 psi, and TEM set to 500°C.

[0047] The second step is the testing of actual samples

[0048] Add the sample to a solution of N,N-diethylphenylalanine succinimide ester of known concentration (the actual sample must be centrifuged to remove the precipitate and filtered through a 0.25 μm filter membrane) and allow to derivatize for 10 minutes to obtain the sample solution to be tested.

[0049] A certain volume of internal standard solution (i.e., a standard solution of N,N-dimethylphenylalanine succinimidyl ester-amino acid with a known concentration) is added to the sample solution to be tested. The mixture of the sample solution to be tested and the internal standard solution is then qualitatively and quantitatively analyzed using LC-MRM. The amino acid mass spectrometry parameters are used for qualitative analysis, and the amino acid content in the sample is calculated based on the standard curve corresponding to the amino acid and the internal standard concentration.

[0050] Among them, the separation spectra of the above 51 amino acids are shown in Figure 1 .Depend on Figure 1 It can be seen that the present invention can achieve the resolution of the remaining amino acids except D / L-Orn, D / L-cis-4-OH-Pro, D-Leu / D-Ile, and D / L-trans-4-OH-Pro and the quantification of all 51 amino acids.

[0051] Example 4 Reliability Analysis of the Detection Method Constructed in Example 3 of the Present Invention

[0052] 1. The standard curve, LOD and LOQ of amino acids in Example 3 are shown in Table 1.

[0053] Table 1 Standard curves, LODs, and LOQs of amino acids

[0054]

[0055] As shown in Table 1, the above amino acids have a good linear relationship in the range of 4-20000 pmol / ml, and the correlation coefficient R 2 All of them are above 0.9938, indicating that the relative quantitative results of the detection method of the present invention are good.

[0056] As shown in Table 1, the LOD (limit of detection) for the 43 amino acids ranged from 0.062 to 7.742 fmol, and the LOQ (limit of quantification) ranged from 0.258 to 25.806 fmol. The detection method of the present invention has low detection limits and quantification limits, indicating that the detection method of the present invention has high sensitivity, accuracy, and precision.

[0057] 2. Amino acid standard solutions of three different concentrations (low, medium, and high) were tested according to the detection method in Example 3, and the accuracy of each amino acid was calculated. As shown in Table 2, the accuracy of the amino acids in the table ranged from 85% to 114.27%, indicating that the present invention has a high accuracy.

[0058] Table 2 Accuracy of amino acids

[0059]

[0060] 3. Precision

[0061] The precision of each amino acid is expressed as the relative standard deviation (RSD). An RSD value below 10% indicates good precision. The mixed amino acid standard solution was analyzed three times on the same day to determine intra-day precision; the mixed amino acid standard solution was tested on three consecutive days to calculate inter-day precision. As shown in Table 3, the RSD values ​​for the amino acids were all below 10%, indicating good precision.

[0062] Table 3 Precision of amino acids

[0063]

[0064] 4. Stability

[0065] Low, medium, and high concentration mixed amino acid standard solutions were stored at 4°C for two days and then analyzed according to the detection method in Example 3. The mixed amino acid standard solution was stored at -20°C for five days and then analyzed according to the detection method in this example. The coefficient of variation (CV) value for each amino acid was calculated and determined. As shown in Table 4, the CV value for each amino acid was less than 8%, indicating that the present invention has good stability.

[0066] Table 4 Stability of amino acids

[0067]

[0068] In summary, the present invention uses the derivative internal standard method to achieve the separation of 43 amino acids and the quantification of 51 amino acids, with high sensitivity, accuracy, precision and stability. It can be used for the detection of D / L-amino acids in complex samples and provides a new detection idea for the separation of D / L-amino acids.

Claims

1. A non-stable isotope-labeled amino acid derivatization reagent pair, characterized in that: The amino acid derivatization reagent pair is composed of N,N-dimethylphenylalanine succinimide ester and N,N-diethylphenylalanine succinimide ester, and the structural formulas of N,N-dimethylphenylalanine succinimide ester and N,N-diethylphenylalanine succinimide ester are respectively: ； 。 2. A method for preparing the amino acid derivatization reagent pair based on non-stable isotope labeling according to claim 1, characterized in that: The preparation method comprises the following contents: L-phenylalanine and paraacetaldehyde or paraformaldehyde are mixed together, TFE and NaBH4 are added, and the mixture is stirred under reflux conditions. After the reaction is completed, N,N-diethylphenylalanine or N,N-dimethylphenylalanine is obtained by recrystallization using methanol-acetone. In N,N-dimethylformamide as a solvent, N,N-diethylphenylalanine or N,N-dimethylphenylalanine is reacted with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at room temperature with stirring; after the reaction is clear, N,N-diethylphenylalanine succinimide ester or N,N-dimethylphenylalanine succinimide ester is purified to obtain a white powder.

3. Use of the amino acid derivatization reagent pair according to claim 1 or the amino acid derivatization reagent pair prepared according to claim 2 in the resolution of chiral amino acids for purposes other than disease diagnosis, wherein: The application uses amino acids derivatized with N,N-dimethylphenylalanine succinimide ester as internal standards.

4. The use according to claim 3, characterized in that: The application includes the following detection steps: first, adding N,N-diethylphenylalanine succinimide ester to the sample for derivatization reaction; In the second step, LC-MRM is used to perform qualitative separation and quantitative detection on the derivatized test solution. The quantitative detection includes the following steps: derivatizing amino acid standards with N,N-diethylphenylalanine succinimidyl ester and N,N-dimethylphenylalanine succinimidyl ester, respectively, and mixing the derivatized amino acids at a certain volume ratio to obtain mixed derivatized amino acids with different concentration ratios; LC-MRM was used to detect and analyze mixed derivatized amino acids at different concentration ratios. A standard curve was drawn with the concentration ratio of N,N-diethylphenylalanine succinimide ester-amino acid and N,N-dimethylphenylalanine succinimide ester-amino acid as the abscissa and the area ratio of N,N-diethylphenylalanine succinimide ester-amino acid and N,N-dimethylphenylalanine succinimide ester-amino acid as the ordinate. A known concentration of N,N-dimethylphenylalanine succinimidyl ester-amino acid was added to the derivatized sample as an internal standard solution, and the sample was detected and analyzed using LC-MRM. The amino acid mass spectrometry parameters were used for qualitative analysis, and the amino acid content in the sample was determined based on the standard curve and the internal standard. The liquid chromatography conditions for LC-MRM were as follows: mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, and the elution conditions were: 10%-90% B from 0 to 23 min. The mass spectrometry conditions were as follows: ESI source in positive ion mode, multiple reaction monitoring scan mode, ISVF set to 5500 V, GS1 set to 55 psi, GS2 set to 55 psi, CUR set to 25 psi, and TEM set to 500 °C.

5. The use according to claim 4, characterized in that: The amino acids in the internal standard solution include D / L-Ala, D / L-Val, D / L-Leu, D / L-Ile, D / L-Met, D / L-Pro, D / L-Trp, D / L-Ser, D / L-Tyr, D / L-Phe, D / L-Asn, D / L-Gln, D / L-Thr, D / L-Asp, D / L-Glu, D / L-Lys, D / L-Arg, D / L-His, D / L-Homoser, D / L-Cit, D / L-Kynu, D / L-Abu and Gly.

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