Method for detecting amniotic fluid organic acid and amino acid metabolites

Abstract

The application discloses a detection method of amniotic fluid organic acid and amino acid metabolites, which comprises the following steps: taking an amniotic fluid sample, adding an internal standard solution, adding hydroxylamine hydrochloride solution and sodium hydroxide solution, mixing, and placing at room temperature for 1 hour for oximation reaction, then adding hydrochloric acid solution for acidification, adding an extraction solvent composed of ethyl acetate and methanol for extraction, centrifuging, taking supernatant, blowing dry with nitrogen, adding a silylating reagent for incubation for derivatization reaction, and finally performing gas chromatography-mass spectrometry detection. The detection method can effectively extract polar organic acids in amniotic fluid, the method reduces emulsification and matrix effect, and the detection result is excellent in stability; through optimization of pretreatment, trace organic acids 2-ketoisovaleric acid, 2-keto-3-methylvaleric acid, fumaric acid, malic acid, 2-ketoglutaric acid and aconitic acid in amniotic fluid can be accurately detected, the method is high in sensitivity, and through excellent sensitivity and stability, the detection accuracy is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for detecting organic acids and amino acid metabolites in amniotic fluid. Background Technology

[0002] Amniotic fluid is the direct environment for fetal growth and development, and its metabolites can reflect the fetal metabolic state to a certain extent. Organic acid metabolism disorders are common inherited metabolic diseases that can lead to fetal developmental abnormalities. Therefore, establishing accurate and sensitive methods for detecting organic acids in amniotic fluid is of great significance for prenatal diagnosis.

[0003] Currently, the detection of organic acids mainly relies on GC-MS technology, especially urinary organic acid detection, which is a common and important method for the clinical diagnosis of organic aciduria. However, amniotic fluid samples have a complex matrix, containing a large amount of proteins, salts, and other endogenous interfering substances, making pretreatment quite difficult. Existing pretreatment methods are mainly for urine, and when directly applied to amniotic fluid, they suffer from problems such as low extraction efficiency or inability to extract, severe interference from impurities, and insufficient sensitivity.

[0004] Chinese patent CN03118797.8 discloses a method for detecting urinary organic acid / amino acid metabolites using filter paper gas chromatography-mass spectrometry, which employs a two-stage extraction with ethyl acetate and diethyl ether. However, when this method is applied to amniotic fluid, the high protein content leads to severe emulsification during ethyl acetate extraction, making it impossible to properly separate the organic phase after centrifugation, resulting in extremely low recovery rates. Currently, no relevant Chinese patents mention methods for extracting and detecting organic acids from amniotic fluid. Summary of the Invention

[0005] To address the challenges of pretreatment in existing methods for detecting organic acids in amniotic fluid due to the complex matrix of the sample, containing abundant proteins, salts, and other endogenous interfering substances, this invention proposes a method for detecting organic acids and amino acid metabolites in amniotic fluid. This method boasts high extraction efficiency, excellent purification effect, and high sensitivity.

[0006] The objective of this invention is achieved through the following technical solution: A method for detecting organic acids and amino acid metabolites in amniotic fluid, characterized in that: (1) Sample pretreatment: Take amniotic fluid sample, add internal standard solution, mix well. The internal standard solution contains ethyl acetate solution of heptadecanoic acid, C24 alkyl and tropine. (2) Oxime reaction: Add hydroxylamine hydrochloride solution and sodium hydroxide solution to the mixture in step (1), mix well and let stand at room temperature for 1 hour to carry out the oxime reaction; (3) Acidification extraction: Add hydrochloric acid solution to the system after the oxime reaction for acidification, then add amniotic fluid extraction solvent for extraction by vortexing and centrifugation, and take the supernatant after vortexing and mixing. (4) Concentration and derivatization: Add anhydrous sodium sulfate to the supernatant in step (3) to remove excess water, blow the supernatant dry with nitrogen, add silanizing reagent MSTFA + 1% TMCS and incubate at 80℃ for 30 minutes to carry out derivatization reaction; (5) GC-MS detection: The derivatized product was detected by gas chromatography-mass spectrometry.

[0007] Furthermore, in step (1), the concentration of heptadecanoic acid in the internal standard solution is 0.4~0.6 mg / L, the concentration of C24 alkyl (C24) is 0.4~0.6 mg / mL, and the concentration of tropine is 0.8~1.2 mg / mL.

[0008] Furthermore, the volume ratio of the amniotic fluid sample to the internal standard solution is 1:0.015~0.025.

[0009] Furthermore, in step (2), the volume ratio of hydroxylamine hydrochloride solution, sodium hydroxide solution and internal standard solution in step (1) is 25:20:1.5~2.5, the concentration of hydroxylamine hydrochloride solution is 45~55 g / L, and the concentration of sodium hydroxide solution is 95~105 g / L.

[0010] Furthermore, the concentration of the hydrochloric acid solution in step (3) is 5.5~6.5 mol / L, and the volume ratio of the hydrochloric acid solution to the internal standard solution in step (1) is 35:3~5.

[0011] Furthermore, the extraction solvent in step (3) is a mixture of ethyl acetate and methanol in a volume ratio of 7.8:2.2 to 8.2:1.8.

[0012] Furthermore, the GS-MS detection is as follows: The derivatized solution was injected for analysis. Chromatographic conditions: column type and specifications: DB-5, 30m×0.25mm×1.00μm; temperature program: 100℃ for 4 minutes, then increased to 280℃ at a rate of 4℃ / min and held for 11 minutes. Mass spectrometry conditions: Ionization mode (EI); Inlet temperature 280℃; Ion source temperature 200℃; Interface temperature 280℃; Flow control mode: constant linear velocity (43.0 cm / sec); Scan mode: full scan mode; Mass range: 50-500 amu.

[0013] The sample analysis time is 1 hour. The computer analysis software records the chromatographic and mass spectrometric graphs to obtain the corresponding chromatograms.

[0014] Each chromatographic peak was identified by comparison with a mass spectrometry library, and the abundance of each peak was qualitatively and quantitatively analyzed.

[0015] The present invention has the following technical effects: The detection method of this invention can effectively extract polar organic acids from amniotic fluid, especially significantly improving the response intensity of glycolic acid, 3-hydroxybutyric acid, phosphoric acid, citric acid, palmitic acid, heptadecanic acid, octadecanoic acid, octadecanoic acid, and C24 alkyl. This detection system can effectively reduce emulsification and matrix effect, and the detection results have excellent stability. Through optimized pretreatment, trace amounts of organic acids such as 2-ketoisovaleric acid, 2-keto-3-methylvaleric acid, fumaric acid, malic acid, 2-ketoglutaric acid, and aconitic acid in amniotic fluid can be accurately detected with high sensitivity. Through excellent sensitivity and stability, the detection accuracy is significantly improved. Attached Figure Description

[0016] Figure 1 The present invention provides a chromatogram of organic acid analysis in amniotic fluid from normal pregnant women using a detection method.

[0017] Figure 2 The present invention provides a chromatogram of organic acid analysis in the amniotic fluid of pregnant women with abnormalities, obtained using the detection method of the present invention.

[0018] Figure 3 Chromatograms corresponding to different extraction solvent treatment groups. Detailed Implementation

[0019] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0020] Example 1 A method for detecting organic acids and amino acid metabolites in amniotic fluid, characterized in that: (1) Sample pretreatment: Take 2 ml of amniotic fluid sample from the second trimester, add 40 μl of internal standard solution, mix well. The internal standard solution contains ethyl acetate of heptadecanoic acid (0.5 mg / ml), C24 alkyl (C24, 0.5 mg / ml) and troponic acid (1.0 mg / ml); (2) Oxime reaction: Add 500 μL of hydroxylamine hydrochloride solution with a concentration of 50 g / L and 400 μL of sodium hydroxide solution with a concentration of 100 g / L to the mixture in step (1), mix well and let stand at room temperature for 1 hour to carry out the oxime reaction. (3) Acidification extraction: Add 350 μl of hydrochloric acid solution to the system after the oxime reaction for acidification, then add 7 ml of amniotic fluid extraction solvent for extraction by vortexing and shaking. After vortexing and mixing, centrifuge at 4000 rpm and take the supernatant. The extraction solvent is a mixture of ethyl acetate and methanol in a volume ratio of 8:2. (4) Add 1.5 mg of anhydrous sodium sulfate to the supernatant in step (3) to remove excess water; (5) Concentration and derivatization: Add 1.5 mg of anhydrous sodium sulfate to the supernatant to remove excess water, blow the supernatant dry with nitrogen, add 100 μl of silanizing reagent MSTFA and 1% of the mass of TMCS derivatizing reagent MATFA, and incubate at 80 °C for 30 minutes to carry out the derivatization reaction. (6) GC-MS detection: The derivatized product was detected by gas chromatography-mass spectrometry. The derivatized solution was injected for analysis. Chromatographic conditions: Column type and specifications: DB-5, 30m×0.25mm×1.00μm; Temperature program: 100℃ for 4 minutes, then increase to 280℃ at a rate of 4℃ / min and hold for 11 minutes.

[0021] Mass spectrometry conditions: Ionization mode (EI); Inlet temperature 280℃; Ion source temperature 200℃; Interface temperature 280℃; Flow control mode: constant linear velocity (43.0 cm / sec); Scan mode: full scan mode; Mass range: 50-500 amu.

[0022] The sample analysis time is 1 hour. The computer analysis software records the chromatographic and mass spectrometric graphs, identifies each chromatographic peak by comparing it with the mass spectrometry library, and performs qualitative and quantitative analysis on the abundance of each chromatographic peak.

[0023] The above methods were used to analyze amniotic fluid from normal pregnant women and amniotic fluid from abnormal pregnant women (prenatal diagnosis of suspected D-2-hydroxyglutaric aciduria type I in the fetus). D2HGDH Organic acids (complex heterozygous variants) were detected. The chromatogram of organic acid analysis in normal amniotic fluid is shown below. Figure 1 As shown, the chromatographic peaks in the figure were identified in sequence as follows: 1 (lactic acid), 2 (glycolic acid), 3 (oxalic acid), 4 (pyruvic acid), 5 (3-hydroxybutyric acid), 6 (2-hydroxy-3-methylbutyric acid), 7 (2-ketoisovaleric acid), 8 (urea), 9 (phosphate), 10 (2-ketoisohexanoic acid), 11 (succinic acid), 12 (glyceric acid), 13 (fumaric acid), 14 (2-hydroxypropyl-2-hydroxypropionic acid), 15 (malic acid), 16 (L-5-oxoproline), 17 (tropine acid), 18 (2-ketoglutarate), 19 (aconitic acid), 20 (citric acid-3TMS), 21 (citric acid-4TMS), 22 (hippuric acid), 23 (palmitic acid), 24 (heptadecanic acid), 25 (linolenic acid), 26 (octadecanoic acid), 27 (octadecanoic acid), 28 (arachidonic acid), and 29 (C24 alkyl).

[0024] Chromatogram of organic acid analysis in amniotic fluid of pregnant women with abnormalities, as shown below Figure 2As shown, the chromatographic peaks in the figure were identified in sequence as follows: 1 (lactic acid), 2 (glycolic acid), 3 (oxalic acid), 4 (pyruvic acid), 5 (3-hydroxybutyric acid), 6 (2-hydroxy-3-methylbutyric acid), 7 (2-ketoisovaleric acid), 8 (phosphate), 9 (2-ketoisohexanoic acid), 10 (succinic acid), 11 (glyceric acid), 12 (fumaric acid), 13 (2-hydroxypropyl-2-hydroxypropionic acid), 14 (malic acid), 15 (L-5-oxoproline), 16 (2-hydroxyglutaric acid), 17 (tropine acid), 18 (2-ketoglutaric acid), 19 (aconitic acid), 20 (citric acid-4TMS), 21 (hippuric acid), 22 (palmitic acid), 23 (heptadecanic acid), 24 (octadecanoic acid), 25 (octadecanoic acid), and 26 (C24 alkyl).

[0025] Using this method, 2-hydroxyglutaric acid was successfully detected in amniotic fluid, which was significantly higher than that in normal amniotic fluid, and successfully assisted in the prenatal diagnosis of a case of D-2-hydroxyglutaric aciduria type I.

[0026] To verify the advantages of the extraction solvent in this invention, a corresponding comparative experiment was conducted.

[0027] Control group 1: The same conditions as in Example 1 were used, except that the extraction solvent was replaced with pure ethyl acetate.

[0028] Control group 2: The same conditions as in Example 1 were used, except that the extraction solvent was replaced with ethyl acetate:methanol = 9:1.

[0029] Control group 3: The same conditions as in Example 1 were used, except that the extraction solvent was replaced with ethyl acetate:methanol = 7:3.

[0030] Pretreatment and detection were performed using the three methods described above. Heptadecanic acid was used as an internal standard. The spiked recoveries of tropine and C24 alkyl were calculated, and the results are shown in Table 1.

[0031] Table 1: Effect of different extraction solvents on recovery rate

[0032] As shown in Table 1, the 8:2 ratio of mixed solvent used in this invention has the closest extraction recovery rate of tropinic acid from amniotic fluid to the theoretical value. In comparison, the recovery rates of other ratios of extracts have larger errors compared to the theoretical values, indicating that the technical solution of this invention has achieved unexpected technical effects.

[0033] Typically, effective protein precipitation with methanol requires extremely high concentrations, and the protein precipitation and extraction steps must be performed separately. However, this invention achieves excellent extraction results by mixing ethyl acetate and methanol at a relatively low methanol concentration. This indicates that methanol in this invention does not act as a protein precipitator, but rather optimizes the physicochemical properties of the extraction system. The addition of methanol fine-tunes the polarity of ethyl acetate and reduces the interfacial tension between the aqueous and organic phases, which is beneficial for detecting substance transfer from the aqueous to the organic phase and reduces emulsification.

[0034] The chromatograms obtained after treatment in Example 1 and Control Groups 1 and 2 are shown below. Figure 3 As shown in the figure (black represents Example 1, blue represents Control Group 1, and red represents Control Group 2), the organic acids such as glycolic acid, propionic acid, pyruvic acid, 3-hydroxybutyric acid, and phosphoric acid showed the strongest response intensity when treated with the extract of Example 1 at a ratio of 8:2. Tropine acid and citric acid also showed the strongest response intensity when treated with the extract of Example 1. Furthermore, palmitic acid, heptadecanic acid, octadecanoic acid, octadecanoic acid, and C24 alkyl were treated with the extract at a ratio of 8:2. The figure demonstrates that the 8:2 mixed solvent used in this invention to extract organic acids from amniotic fluid resulted in the strongest response intensity for most organic acids, with tropine acid showing the closest recovery rate to the theoretical value, indicating the most ideal detection results.

[0035] Example 2 Methodological validation examines the precision and accuracy of the detection method in Example 1. Precision is determined by repeatedly analyzing the same sample and calculating the coefficient of variation (CV). A good and stable detection method should have a CV of less than 15%. Accuracy is determined by adding a known amount of standard to a real sample (spiking recovery experiment) and calculating the recovery rate. The results are shown in Table 2.

[0036] Table 2: Intra-batch precision and accuracy of each test indicator

[0037] Note: The mean N above is the average ratio of the peak area of ​​the target compound to the peak area of ​​the internal standard heptadecanoic acid, so there is no specific unit; due to different test batches, the recovery rates in Table 2 differ from those in Table 1.

[0038] Table 3. Inter-batch precision of each detection indicator

[0039] As can be seen from Tables 2 and 3, the intra-batch and inter-batch precision results obtained by this method are excellent.

Claims

1. A method for detecting organic acids and amino acid metabolites in amniotic fluid, characterized in that, Follow these steps: (1) Sample pretreatment: Take amniotic fluid sample, add internal standard solution, mix well. The internal standard solution contains ethyl acetate solution of heptadecanoic acid, C24 alkyl and tropine. (2) Oxime reaction: Add hydroxylamine hydrochloride solution and sodium hydroxide solution to the mixture in step (1), mix well and let stand at room temperature for 1 hour to carry out the oxime reaction; (3) Acidification extraction: Add hydrochloric acid solution to the system after the oxime reaction for acidification, then add amniotic fluid extraction solvent for extraction by vortexing and centrifugation, and take the supernatant after vortexing and mixing. (4) Concentration and derivatization: Add anhydrous sodium sulfate to the supernatant in step (3) to remove excess water, blow the supernatant dry with nitrogen, add silanizing reagent MSTFA and incubate at 80℃ for 30 minutes to carry out derivatization reaction; (5) GC-MS detection: The derivatized product was detected by gas chromatography-mass spectrometry.

2. The method for detecting organic acids and amino acid metabolites in amniotic fluid as described in claim 1, characterized in that: In step (1), the concentration of heptadecanoic acid in the internal standard solution is 0.4~0.6 mg / L, the concentration of C24 alkyl (C24) is 0.4~0.6 mg / mL, and the concentration of tropine is 0.8~1.2 mg / mL.

3. The method for detecting organic acids and amino acid metabolites in amniotic fluid as described in claim 1 or 2, characterized in that: The volume ratio of the amniotic fluid sample to the internal standard solution was 1:0.015~0.

025.

4. A method for detecting organic acids and amino acid metabolites in amniotic fluid as described in any one of claims 1-3, characterized in that: The volume ratio of hydroxylamine hydrochloride solution, sodium hydroxide solution and internal standard solution in step (2) is 25:20:1.5~2.5, the concentration of hydroxylamine hydrochloride solution is 45~55 g / L, and the concentration of sodium hydroxide solution is 95~105 g / L.

5. The method for detecting organic acids and amino acid metabolites in amniotic fluid as described in claim 4, characterized in that: The concentration of the hydrochloric acid solution in step (3) is 5.5~6.5 mol / L, and the volume ratio of the hydrochloric acid solution to the internal standard solution in step (1) is 35:3~5.

6. The method for detecting organic acids and amino acid metabolites in amniotic fluid as described in claim 5, characterized in that: The solvent used in step (3) is a mixture of ethyl acetate and methanol in a volume ratio of 7.8:2.2 to 8.2:1.

8.

7. The method for detecting organic acids and amino acid metabolites in amniotic fluid as described in claim 6, characterized in that: The GS-MS detection is as follows: The derivatized solution was injected for analysis. Chromatographic conditions: column type and specifications: DB-5, 30m×0.25mm×1.00μm; temperature program: 100℃ for 4 minutes, then increased to 280℃ at a rate of 4℃ / min and held for 11 minutes. Mass spectrometry conditions: Ionization mode (EI); Inlet temperature 280℃; Ion source temperature 200℃; Interface temperature 280℃; Flow control mode: constant linear velocity (43.0 cm / sec); Scan mode: full scan mode; Mass range: 50-500 amu. The sample analysis time is 1 hour. The computer analysis software records the chromatographic and mass spectrometric graphs to obtain the corresponding chromatograms.

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