Joint detection kit for piperidine acid and 6-oxopiperidinecarboxylic acid as well as detection method and application of joint detection kit

Abstract

The invention provides a joint detection kit for piperidine acid and 6-oxopiperidinecarboxylic acid as well as a detection method and application of the joint detection kit. The kit comprises an internal standard solution containing internal standard substances at 2-site and 5-site deuterated hydrogen substitution positions of a piperidine ring, a precipitator and the like. The detection method comprises the following steps: adding an internal standard solution and a precipitator into a to-be-detected sample to remove protein, dissolving and carrying out other steps, and carrying out liquid chromatography detection. The detection accuracy and sensitivity are improved, the operation is simple and convenient, and the stability and repeatability are good. According to the present invention, the kit can accurately and quantitatively detect piperidine acid and 6-oxopiperidinecarboxylic acid in blood and urine, provides reliable support for prediction, screening and diagnosis of pyridoxine-dependent epilepsy, overcomes the disadvantages and defects of the existing detection method, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis and detection, and particularly relates to a kit for the combined detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, a detection method thereof, and an application thereof. Background Art

[0002] In the field of medical detection, accurately detecting the content of specific substances in the body is crucial for the diagnosis, treatment, and research of diseases. The metabolism of pipecolic acid and 6-oxopiperidinecarboxylic acid in the body is closely related to pyridoxine-dependent epilepsy. Pyridoxine-dependent epilepsy is a rare genetic metabolic disease with a complex pathogenesis. Studies have found that the abnormal metabolism of pipecolic acid and 6-oxopiperidinecarboxylic acid in patients is closely related to the occurrence and development of this disease. By accurately detecting the content of pipecolic acid and 6-oxopiperidinecarboxylic acid in blood and urine, important clues can be provided for the prediction and screening of pyridoxine-dependent epilepsy.

[0003] Currently, there are many deficiencies in the detection methods for pipecolic acid and 6-oxopiperidinecarboxylic acid. Traditional detection methods are often complex in operation, with low detection accuracy and sensitivity, and are difficult to meet the needs of clinical diagnosis and research. For example, in the existing detection process, the accuracy of the detection results may be affected due to the lack of a suitable internal standard. At the same time, the reagents and standards used in the detection method may not be optimized enough to effectively separate and quantify the substances to be detected. In addition, problems such as incomplete protein removal and poor derivatization treatment may exist in the sample processing of the existing methods, affecting the reliability of the detection results.

[0004] Therefore, there is an urgent need to find a detection method for the combined detection of pipecolic acid and 6-oxopiperidinecarboxylic acid that is simple in operation, high in accuracy, and high in sensitivity, and to find the most suitable standard and internal standard, and develop a corresponding kit for the combined detection of pipecolic acid and 6-oxopiperidinecarboxylic acid to overcome the deficiencies and defects of the existing methods and ensure the accuracy and stability of the detection results. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a combined detection kit for pipecolic acid and 6-oxopiperidinecarboxylic acid, its detection method and application. By precisely synthesizing the internal standard 6-oxopiperidine-2-carboxylic acid-D3 with deuterated hydrogen substituted at the 2-position and 5-position of the piperidine ring and applying it to the kit, it can effectively calibrate the matrix effect during the detection process and reduce the influence of interference factors on the detection results. At the same time, the optimized combination of the internal standard solution and the precipitant in the kit can efficiently remove protein impurities in the sample and convert the substance to be detected into a form more suitable for detection. In the detection method, the operating conditions of each step are strictly controlled, such as the volume ratio of the internal standard solution to the precipitant, etc., ensuring the accuracy and repeatability of the detection. At the same time, it improves the accuracy and sensitivity of the detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, can accurately quantify the substance to be detected in complex biological samples, and provides reliable data support for the diagnosis and research of diseases; and the operation is simple, reducing the operation time and labor cost, improving the detection efficiency; it also has good stability and repeatability, reducing the risk of operation errors and ensuring the stability of the product quality. It provides an efficient, accurate and reliable solution for the combined detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, and has important application value especially in the prediction, screening and diagnosis of pyridoxine-dependent epilepsy.

[0006] On the one hand, the present invention provides an internal standard for 6-oxopiperidinecarboxylic acid, and the internal standard is 6-oxopiperidine-2-carboxylic acid-D3. In the internal standard, the substitution positions of deuterated hydrogen include one or more of the following: hydrogen substitution at the 2-position, 3-position, 4-position, 5-position of the piperidine ring, or hydrogen substitution on the carbon atom to which the formic acid group is attached.

[0007] In the present invention, 6-oxopiperidine-2-carboxylic acid-D3 (D3-OXO-PIP) is the internal standard for 6-oxopiperidine-2-carboxylic acid, and 6-oxopiperidine-2-carboxylic acid is 6-oxopiperidinecarboxylic acid (6-OXO-PIP).

[0008] Furthermore, in the internal standard, the substitution positions of deuterated hydrogen include hydrogen substitution at the 2-position and 5-position of the piperidine ring; the structural formula of 6-oxopiperidine-2-carboxylic acid-D3 is as follows:

[0009]

[0010] With the continuous development of medical detection technologies, the demand for accurate detection of specific substances in organisms is increasing. Especially in fields related to metabolic diseases, such as research on pyridoxine-dependent epilepsy, the accurate detection of 6-oxopiperidinecarboxylic acid is of great significance. However, existing internal standards face many challenges in practical applications. Traditional methods for synthesizing internal standards often have difficulty controlling the substitution positions of deuterated hydrogens, leading to an increase in the structural uncertainty of the internal standard and making it difficult to ensure similarity with the substance to be measured, thereby affecting the accuracy and reliability of detection.

[0011] Through in-depth theoretical research and extensive experimental exploration, the present invention has determined 6-oxopiperidine-2-carboxylic acid-D3 as an internal standard, and has clarified that internal standards 6-oxopiperidine-2-carboxylic acid-D3 with different deuterated hydrogen substitution positions can be prepared by different synthesis methods. The specific substitution positions include one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th, and 5th positions of the piperidine ring, or hydrogen substitution on the carbon atom to which the formic acid group is attached. By precisely controlling the substitution positions of deuterated hydrogens, the physicochemical properties of the internal standard can be adjusted to make it closer to the substance to be measured, thereby improving the accuracy and reliability of detection.

[0012] Therefore, in order to prepare 6-oxopiperidine-2-carboxylic acid-D3 with the best performance in the detection of piperidine acid (PA) and 6-oxopiperidinecarboxylic acid (6-OXO-PIP) in the present invention, different synthesis methods were screened in some embodiments. Initially, due to insufficient understanding of the relationship between molecular structure and detection performance, the design of the internal standard lacked pertinence, resulting in problems such as poor stability, low matching degree with the substance to be measured, and low detection accuracy in the detection of piperidine acid and 6-oxopiperidinecarboxylic acid. Therefore, systematic experimental studies were carried out, including changing factors such as reaction temperature, time, and solvent system, to determine the optimal synthesis conditions and deuterated hydrogen substitution positions. During the experiment, the possibilities of different deuterated hydrogen substitution positions were explored one by one, and various deuterated combinations at different positions of the piperidine ring and on the carbon atom to which the formic acid group is attached were tried by different synthesis methods. Through comparative analysis of a large amount of experimental data, it was finally found that 6-oxopiperidine-2-carboxylic acid-D3 prepared with deuterated hydrogen substitution positions at the 2nd and 5th positions of the piperidine ring can maintain stable performance in the detection environment of piperidine acid and 6-oxopiperidinecarboxylic acid.

[0013] This is because the deuterated hydrogen substitution positions at the 2- and 5-positions of the piperidine ring enable the internal standard to play an excellent role in the detection of complex biological samples. On the one hand, its structure is more similar to the analyte, which can effectively calibrate the matrix effect during the detection process, thereby not only improving the accuracy and precision of the detection of pipecolic acid and 6-oxopiperidinecarboxylic acid. During chromatographic analysis, the internal standard with deuterated hydrogen substitution at the 2- and 5-positions has similar polarity and intermolecular forces to the analyte, making their retention behaviors on the chromatographic column highly consistent. This ensures that in the presence of a complex biological matrix, the internal standard can accurately track the elution process of the analyte, reducing problems such as peak tailing and peak area changes caused by the interaction between matrix components and the analyte, and thus improving the accuracy of quantitative analysis. On the other hand, this specific substitution position endows the internal standard with better chemical stability. From the perspective of molecular structure, the deuterated hydrogen substitution at the 2- and 5-positions does not cause a drastic change in the intramolecular electron cloud distribution, maintaining the overall stability of the molecule. It can maintain stable properties under different conditions such as temperature and pH. In a high-temperature environment, it is not prone to decomposition or structural rearrangement reactions, ensuring reliability in various experimental conditions and actual detection scenarios. In an acidic or basic environment, its chemical structure can remain intact and will not change its similarity and calibration ability with the analyte due to the protonation or deprotonation process, thus ensuring stable performance in different sample systems. In addition, this substitution position also endows the internal standard with good stability and repeatability. During long-term storage, the internal standard with deuterated hydrogen substitution at the 2- and 5-positions is not prone to degradation or deterioration, and can maintain the consistency of its physicochemical properties, reducing the detection errors introduced by the changes in the internal standard itself. In multiple repeated experiments, its response relationship with the analyte always remains stable, making the detection results of different batches highly comparable and repeatable, providing reliable technical support for the accurate detection of pipecolic acid and 6-oxopiperidinecarboxylic acid in vivo and the diagnosis and research of related diseases, greatly enhancing the reliability and practicality of the detection method, and strongly promoting the research progress and clinical application accuracy in related fields.

[0014] On the other hand, the present invention provides a method for preparing an internal standard, wherein the internal standard is as described in any one of the above technical solutions; the internal standard is obtained by reacting 6-oxopiperidine-2-carboxylic acid as a raw material, and includes the following synthetic route:

[0015]

[0016] Further, the preparation method includes the following steps:

[0017] (1) Using 6-oxopiperidine-2-carboxylic acid as a raw material, adding a catalytic solvent, and heating to obtain a reaction mixture solution;

[0018] (2) Cool the mixed solution obtained in step (1) to room temperature, then concentrate, extract. The organic phase is dried, filtered, and then concentrated under reduced pressure and subjected to column chromatography to obtain 6-oxopiperidine-2-carboxylic acid-D3.

[0019] In some embodiments, in order to further improve the efficiency, purity and stability of D3-OXO-PIP synthesis in the prior art, different synthesis methods were screened in order to prepare D3-OXO-PIP with high purity, high yield, simple operation and low cost. The results show that the yield of Scheme 1 reaches 83%, the purity is 99%, and the synthesis steps are simple, without complex steps such as high-vacuum operation and multiple filtrations; the yield of Scheme 2 is only 71%, the operation is complex and involves harsh conditions such as high-vacuum drying, increasing equipment requirements, operation difficulty and production cost, and may also cause product loss and affect quality stability, restricting large-scale production. From the perspectives of yield, simplicity of operation and purity, Scheme 1 is significantly superior to Scheme 2. In summary, using the best synthesis method (Scheme 1) in Example 1 is the optimal choice of the present invention, which can synthesize the internal standard D3-OXO-PIP with high purity, high yield, simple operation and low cost, providing a reliable synthesis route for detecting specific substances in body fluids such as blood and urine, and improving the detection accuracy and reliability.

[0020] Further, in step (1), the catalytic solvent includes a reaction auxiliary solvent and a basic reaction promoting solvent. The reaction auxiliary solvent includes any one or more of methanol-D1 and methanol, and the basic reaction promoting solvent includes any one or more of NaOD and Na2CO3.

[0021] The reaction auxiliary solvent is used to promote the reaction and increase the solubility of the reactants.

[0022] Further, the dosage of the reaction auxiliary solvent includes 8-12 mL, and the dosage of the basic reaction promoting solvent includes 3.8-4.2 g.

[0023] In some embodiments, in order to further improve the yield and purity of D3-OXO-PIP and improve the stability and repeatability of the synthesis method, so as to reduce costs and improve production efficiency, different reaction conditions in the best synthesis method of D3-OXO-PIP were optimized and screened.

[0024] First, the necessity of the synergistic reaction of adding methanol-D1 and NaOD was experimentally verified. The results showed that only under the synergistic action of methanol-D1 and NaOD could the yield and purity of the internal standard D3-OXO-PIP reach the optimal level. NaOD and methanol-D1 are irreplaceably necessary in the synthesis of the internal standard D3-OXO-PIP in the present invention. This is because in the process of synthesizing 6-oxopiperidine-2-carboxylic acid-D3 from the molecular structure of 6-oxopiperidine-2-carboxylic acid, the basic strength and properties provided by NaOD are required to initiate key reaction steps, such as the activation and substitution process of hydrogen atoms. Without NaOD, these reactions cannot proceed smoothly, and the reactants basically remain unchanged and cannot be converted into the target product. Methanol-D1 can increase the solubility of the reactants in the reaction system, enabling the reactant molecules to better contact and react, thereby improving the reaction efficiency.

[0025] Meanwhile, the optimal dosages of methanol-D1 and NaOD were also optimized. The experimental results showed that it was preferred that the dosage of methanol-D1 was in the range of 8 - 12 mL and the dosage of NaOD was in the range of 3.5 - 4.5 g. At this time, the purity of the product D3-OXO-PIP could reach over 98%. Meanwhile, when methanol-D1 was 10 mL and NaOD was 4 g, the effect was the best.

[0026] Furthermore, in step (1), the heating temperature includes 82 - 88 °C.

[0027] Furthermore, the heating time includes 2.5 - 3.5 days.

[0028] In some embodiments, the reaction temperature was also screened. The experimental results showed that when the temperature was in the range of 82 - 88 °C, the reaction rate was moderate, the side reactions were few, and the synthesis had the best yield and purity. The purity of the internal standard D3-OXO-PIP could reach over 98%. And when the reaction temperature was 85 °C, the yield and purity of the internal standard D3-OXO-PIP reached the best. This may be because within this temperature range, the energy supply of the reaction system was relatively appropriate. It could not only provide sufficient activation energy for the reaction to promote the effective collision and reaction of the reactant molecules, but also would not cause too many side reactions due to excessive energy. For the process of chemical bond breaking and forming involved in this reaction, especially the reaction steps related to deuterium-hydrogen substitution, this temperature range was helpful to maintain the selectivity and high efficiency of the reaction, enabling the target product to be generated in a relatively high proportion, and ensuring the structural integrity and purity of the product.

[0029] When the reaction temperature is too low (80 °C and below), the energy of the reaction system is insufficient, the molecular movement rate slows down, and the effective collision frequency between reactants decreases significantly, resulting in an extremely slow reaction rate. A large amount of reactants fail to participate in the reaction sufficiently, leading to a low yield of the final product. At the same time, due to the extension of the reaction time, the probability of some unnecessary side reactions may increase. These side reactions may introduce impurities or cause partial degradation of the product, thereby affecting the purity of the product. When the reaction temperature is too high (90 °C and above), the excessive energy input makes the reaction system too active and prone to a series of complex side reactions. These side reactions may include processes such as decomposition, isomerization, and unnecessary polymerization of reactants or products. They not only consume a large amount of reactants, reducing the production of the target product, but also generate various impurities that are difficult to separate and remove, seriously affecting the purity of the product. In addition, too high a temperature may have an adverse effect on the chemical equilibrium of the reaction system, disrupting the reaction equilibrium that is originally favorable for the formation of the target product and further reducing the yield and purity of the product.

[0030] Therefore, in the present invention, a reaction temperature of 82 - 88 °C is preferably used to ensure the high efficiency of the synthesis process and the high quality of the product. And when the preferred reaction temperature is 85 °C, the yields and purities of the internal standard D3-OXO-PIP are both optimal.

[0031] In some embodiments, different reaction times are optimized and screened. The experimental results show that when the reaction time is 2.5 - 3.5 days, the best synthesis yields and purities are obtained, and the purities of the internal standard D3-OXO-PIP can all reach over 98%. And when the reaction time is 3 days, the yields and purities of the internal standard D3-OXO-PIP are both optimal. When the reaction time is further reduced or extended, the purity of the internal standard D3-OXO-PIP will be significantly reduced. Therefore, in the present invention, a reaction time of 2.5 - 3.5 days is preferably used to ensure the high efficiency of the synthesis process and the high quality of the product. And when the preferred reaction time is 3 days, the yields and purities of the internal standard D3-OXO-PIP are both optimal.

[0032] Furthermore, in step (2), the eluent for column chromatography includes dichloromethane and methanol; the volume ratio of dichloromethane to methanol is (9:1) - (11:1).

[0033] In some embodiments, the volume ratio of dichloromethane to methanol in the eluent was also optimized and screened. The results showed that when the ratio of V(dichloromethane):V(methanol) in the eluent was 9:1 - 11:1, the best synthesis yield and purity were achieved, and the purity of the internal standard D3-OXO-PIP could reach over 98%. And when the ratio of V(dichloromethane):V(methanol) in the eluent was 10:1, the yield and purity of the internal standard D3-OXO-PIP were both optimal. This is because during the column chromatography process, the ratio of the eluent plays a crucial role in the separation effect of the target product and impurities. Different ratios of dichloromethane and methanol will affect the polarity and elution ability of the eluent. When the ratio is 10:1, the polarity of the eluent is moderate, which can not only ensure sufficient elution ability for the target product D3-OXO-PIP to effectively elute it from the stationary phase, but also avoid over-eluting impurities, thus achieving good separation of the target product and impurities, with the yield reaching 83% and the purity as high as 99.5%. Therefore, in this embodiment, the ratio of V(dichloromethane):V(methanol) in the eluent is preferably 9:1 - 11:1, and when the ratio of the two is 10:1, the yield and purity of the internal standard D3-OXO-PIP are optimal, providing a reliable condition guarantee for the high-quality preparation of the subsequent internal standard.

[0034] On the other hand, the present invention provides a use of an internal standard for preparing a preparation for improving the accuracy, stability, and repeatability of the detection result of 6-oxopiperidine-2-carboxylic acid, characterized in that the internal standard is 6-oxopiperidine-2-carboxylic acid-D3, and the substitution positions of deuterated hydrogen in the internal standard include one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th, and 5th positions of the piperidine ring, or hydrogen substitution on the carbon atom connected to the formic acid group.

[0035] In some embodiments, by applying the internal standard prepared by the present invention to the detection preparation, the matrix effect during the detection process can be effectively calibrated. In a complex biological sample system, various interfering factors will affect the accuracy and stability of the detection result. Due to its specific structure and deuterated hydrogen substitution positions, the internal standard of the present invention can exhibit similar physicochemical properties to the analyte during the detection process, thereby reducing the error caused by matrix interference. The experimental results show that when using the preparation containing this internal standard for detection, the coefficient of variation of the detection results in different samples is significantly reduced, which means that the dispersion degree of the detection results is reduced, and the stability and repeatability are significantly improved. The final results show that the internal standard D3-OXO-PIP prepared by the best synthesis method of the present invention can significantly improve the accuracy, stability, and repeatability of the detection results, and has the effect of providing a reliable guarantee for accurate detection.

[0036] On the other hand, the present invention provides the use of an internal standard for preparing a preparation for improving the detection accuracy and sensitivity of pipecolic acid and 6-oxopiperidinecarboxylic acid in a sample, wherein the internal standard is prepared by the preparation method described in any one of the above technical solutions.

[0037] In some embodiments, for the detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, traditional methods often have problems of insufficient accuracy and sensitivity. The internal standard of the present invention has a highly matched structure with the substance to be detected through a precise synthesis method. During the detection process, it can accurately track and quantify the content changes of pipecolic acid and 6-oxopiperidinecarboxylic acid. Through comparative experiments, it is found that when using the preparation with this internal standard for detection, the target substance can be clearly distinguished from background interference in samples with different concentrations, improving the detection accuracy. At the same time, due to the high purity and stability of the internal standard, it can also effectively play a role in low-concentration samples, thereby improving the detection sensitivity. The final results show that the internal standard D3-OXO-PIP prepared by the optimal synthesis method of the present invention can significantly improve the detection accuracy and sensitivity of pipecolic acid and 6-oxopiperidinecarboxylic acid in a sample, and has the effect of providing accurate data support for the research and diagnosis of related diseases.

[0038] On the other hand, the present invention provides the use of an internal standard for preparing a preparation for predicting whether an individual has pyridoxine-dependent epilepsy, wherein the internal standard is prepared by the preparation method described in any one of the above technical solutions.

[0039] In some embodiments, pyridoxine-dependent epilepsy is a rare genetic metabolic disease, and early diagnosis is difficult. By detecting the contents of pipecolic acid and 6-oxopiperidinecarboxylic acid in the body fluids of patients, important clues can be provided for the diagnosis of the disease. The internal standard of the present invention plays a key role in preparing a preparation for predicting whether an individual has pyridoxine-dependent epilepsy. Through the detection and analysis of a large number of clinical samples, it is found that when specific changes occur in the contents of pipecolic acid and 6-oxopiperidinecarboxylic acid in the patient's body, combined with the detection results of this internal standard, it can accurately determine whether the patient has pyridoxine-dependent epilepsy. This is because the stability and specificity of this internal standard can ensure the reliability of the detection results and reduce the occurrence of false positives and false negatives. The final results show that the internal standard D3-OXO-PIP prepared by the optimal synthesis method of the present invention can be used to prepare a preparation for predicting whether an individual has pyridoxine-dependent epilepsy, and has the effect of providing a powerful tool for early disease diagnosis and intervention.

[0040] On the other hand, the present invention provides a kit for the combined detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, including an internal standard solution. The internal standard solution contains an internal standard substance, 6-oxopiperidine-2-carboxylic acid-D3. In the internal standard substance, the substitution positions of deuterated hydrogen include one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th, and 5th positions of the piperidine ring, or hydrogen substitution on the carbon atom to which the formic acid group is attached.

[0041] The combined detection described in the present invention refers to the simultaneous detection and analysis of pipecolic acid and 6-oxopiperidinecarboxylic acid, and obtaining the content information of the two substances in the sample through one detection process. In medical research and clinical diagnosis, the content changes of pipecolic acid and 6-oxopiperidinecarboxylic acid often have synergy. The combined detection can more comprehensively reflect the metabolic state of the organism and provide a more accurate basis for the diagnosis and treatment of diseases.

[0042] In order to accurately quantitatively detect pipecolic acid and 6-oxopiperidinecarboxylic acid, precise quantification needs to be carried out by the internal standard quantification method. The present invention selects a variety of internal standard substances for pipecolic acid and 6-oxopiperidinecarboxylic acid. The internal standard substances are prepared by different synthesis methods, including 6-oxopiperidine-2-carboxylic acid-D3 with hydrogen substitution at the 2nd and 5th positions of the piperidine ring, and 6-oxopiperidine-2-carboxylic acid-D3 with hydrogen substitution at the 3rd and 5th positions of the piperidine ring. Research has shown that 6-oxopiperidine-2-carboxylic acid-D3 with hydrogen substitution at the 2nd and 5th positions of the piperidine ring prepared by the optimal synthesis method of the present invention is the most suitable internal standard for quantitatively detecting pipecolic acid and 6-oxopiperidinecarboxylic acid. This internal standard substance can exhibit similar physicochemical properties to the substances to be detected during the detection process, thereby enhancing the response signal of the substances to be detected, reducing the error caused by matrix interference, and improving the accuracy of the detection results.

[0043] Furthermore, in the internal standard substance, the substitution positions of deuterated hydrogen include hydrogen substitution at the 2nd position and 5th position of the piperidine ring; the structural formula of 6-oxopiperidine-2-carboxylic acid-D3 is as follows:

[0044]

[0045] On the other hand, the present invention provides a method for the combined detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, using the kit described in any one of the above technical solutions for detection.

[0046] Furthermore, the combined detection method includes the following steps:

[0047] (1) Add the internal standard solution to the sample to be detected, then add a precipitating agent, mix well and centrifuge to separate the supernatant and discard the protein precipitate;

[0048] (2) Take the supernatant from step (1), dry it under nitrogen, and dissolve it with a reconstitution solution to obtain a sample extract;

[0049] (3) Perform liquid chromatography detection on the sample extract.

[0050] Further, in step (1), the precipitant includes any one or a combination of methanol, acetonitrile, acetone, and ethanol.

[0051] Further, in step (1), the volume ratio of the internal standard solution to the precipitant is 1:(2 - 5).

[0052] In some embodiments, the internal standard solution plays an irreplaceable role because it can effectively calibrate the matrix effect during the detection process, ensuring the accuracy and reliability of the detection results. Only when using the internal standard solution 6-oxopiperidine-2-carboxylic acid-D3 can the effects of accurately tracking and quantifying the analytes pipecolic acid and 6-oxopiperidinecarboxylic acid be achieved.

[0053] The precipitant has the function of removing protein impurities from the sample and is indispensable. During the detection process, if protein impurities are not effectively removed, they will interfere with the detection results and reduce the accuracy.

[0054] At the same time, in some ways, a large number of screenings have been carried out on the combination of the internal standard solution and the precipitant. It is found that when the internal standard solution is 6-oxopiperidine-2-carboxylic acid-D3, methanol is preferably used as the precipitant to have the best protein removal and detection accuracy improvement effects. This may be because methanol can rapidly denature and precipitate proteins, and at the same time has good compatibility with the internal standard 6-oxopiperidine-2-carboxylic acid-D3 in the detection system and will not interfere with the detection process.

[0055] And the volume ratio of 6-oxopiperidine-2-carboxylic acid-D3 to methanol is 1:(2 - 5). Within this range, while ensuring the protein removal effect, the calibration function of the internal standard can be fully exerted, having the effects of improving detection sensitivity and accuracy. And when the preferred volume ratio of the two is 1:4, the best detection effect can be achieved, that is, it can maximize the removal of protein impurities, increase the response signal of the analytes, and ensure the accuracy and stability of the detection results.

[0056] In previous studies, the inventors used a derivatization detection method and needed to accurately quantify five substances: piperidine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA), α-aminoadipic acid (AAA), pipecolic acid (PA), and 6-oxopiperidinecarboxylic acid (6-OXO-PIP). Based on this, the preliminary screening and diagnostic evaluation of PDE diseases can be realized. However, this method has significant drawbacks. Its operation process is extremely complex, involving the detection of multiple substances and having extremely high requirements for the control of experimental conditions. This undoubtedly increases the detection cost and time cost, and at the same time increases the probability of detection errors.

[0057] In contrast, the present invention innovatively uses the internal standard method for detection. The main function of the derivatization solution is to chemically modify the target substance, making it easier to be detected and identified in the subsequent detection process, thereby improving the sensitivity and accuracy of the detection. In the present invention, 6-oxopiperidine-2-carboxylic acid-D3 is directly used as the internal standard without going through complex derivatization steps. By virtue of the high consistency of the physical and chemical properties between the internal standard and the analyte to be measured, the matrix effect can be accurately calibrated and the influence of interference factors on the detection result can be reduced.

[0058] Specifically, as the internal standard, 6-oxopiperidine-2-carboxylic acid-D3 has deuterated hydrogen substitution positions (hydrogen substitution at the 2nd and 5th positions of the piperidine ring), which makes it have similar retention behavior and ionization efficiency to the analyte during chromatographic analysis. During the detection process, the internal standard can accurately track the elution process of the analyte, effectively reducing problems such as peak tailing and peak area changes caused by complex matrix components. By using the internal standard method, the present invention not only simplifies the detection process but also significantly improves the accuracy and repeatability of the detection, enabling accurate quantitative detection of piperidine acid and 6-oxopiperidinecarboxylic acid in complex biological samples.

[0059] Therefore, by directly using the internal standard method, the present invention avoids the cumbersome operations and potential errors of the derivatization step, significantly improves the detection efficiency, reduces the detection cost, and provides reliable technical support for the rapid diagnosis and large-scale screening of PDE diseases.

[0060] On the other hand, the present invention provides the use of a composition for preparing a reagent for improving the accuracy and sensitivity of the combined detection of piperidine acid and 6-oxopiperidinecarboxylic acid. The composition includes an internal standard and a precipitant; the internal standard includes 6-oxopiperidine-2-carboxylic acid-D3, and the precipitant includes methanol.

[0061] Further, the internal standard includes 6-oxopiperidine-2-carboxylic acid-D3. In the internal standard, the substitution positions of deuterated hydrogen include one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th, 5th positions of the piperidine ring, or hydrogen substitution on the carbon atom connected to the formic acid group.

[0062] Further, in the internal standard, the substitution positions of deuterated hydrogen include hydrogen substitution at the 2nd and 5th positions of the piperidine ring; the structural formula of 6-oxopiperidine-2-carboxylic acid-D3 is as follows:

[0063]

[0064] Further, the volume ratio of the internal standard to the precipitant is 1:(2 - 5).

[0065] On the other hand, the present invention provides a use of 6-oxopiperidine-2-carboxylic acid-D3 for preparing an internal standard solution for improving the accuracy and sensitivity of the combined detection of pipecolic acid and 6-oxopiperidinecarboxylic acid. In the 6-oxopiperidine-2-carboxylic acid-D3, the substitution positions of deuterated hydrogen include one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th, and 5th positions of the piperidine ring, or hydrogen substitution on the carbon atom to which the formic acid group is attached.

[0066] Further, in the 6-oxopiperidine-2-carboxylic acid-D3, the substitution positions of deuterated hydrogen include hydrogen substitution at the 2nd and 5th positions of the piperidine ring; the structural formula of the 6-oxopiperidine-2-carboxylic acid-D3 is as follows:

[0067]

[0068] Beneficial effects

[0069] 1. A combined detection kit for pipecolic acid and 6-oxopiperidinecarboxylic acid, its detection method and application provided by the present invention. In the kit, a newly synthesized internal standard: 6-oxopiperidine-2-carboxylic acid-D3 is included. By optimizing the synthesis route and conditions, it has high specificity and stability, and has the effect of significantly improving the detection accuracy and reliability. During the synthesis process, the reaction temperature is precisely controlled at 82 - 88 °C, the reaction time is 2.5 - 3.5 days, and the key parameters such as the dosage of NaOD being 3.8 - 4.2 g and the volume ratio of dichloromethane to methanol in the column chromatography eluent being 9:1 - 11:1 are determined to ensure the high-quality production of the internal standard. This high-quality internal standard can closely track the analyte during detection, effectively reducing errors caused by environmental factors and sample matrix differences, making the detection results more stable and reliable, and laying a solid foundation for the accurate diagnosis of diseases.

[0070] 2. The kit and detection method of the present invention have outstanding advantages in terms of operation convenience. The entire detection process is designed to be simple and efficient, with clear steps from sample pretreatment to obtaining the final detection result. In the sample pretreatment stage, the precipitating agent is carefully selected, and the volume ratio of the internal standard solution to the precipitating agent (such as methanol) is optimized to 1:(2 - 5), which can effectively remove protein impurities while fully exerting the calibration function of the internal standard, avoiding complex multi-step operations and cumbersome reagent preparation processes. Operators only need to operate according to the established steps, without special professional skill training, and can successfully complete the detection, greatly shortening the detection time, reducing the labor cost, improving the detection efficiency, and being conducive to wide application in various laboratory and clinical detection scenarios.

[0071] 3. The detection method of the present invention has achieved a significant improvement in detection efficiency. Different from traditional derivatization methods, the detection method of the present invention does not require a derivatization step, but directly uses the internal standard method for detection. The present invention uses 6-oxopiperidine-2-carboxylic acid-D3 as the internal standard substance to effectively calibrate the matrix effect and reduce the influence of interference factors on the detection results. At the same time, it effectively reduces the waiting time and data processing difficulty during the detection process. Traditional derivatization methods require additional chemical reaction steps, which are time-consuming and complex to operate. The internal standard method of the present invention simplifies the detection process and can accurately detect a large number of samples in a short time. This not only improves the detection efficiency but also reduces the risk of operation errors, meeting the requirements for detection timeliness in clinical and scientific research, and providing strong support for the rapid diagnosis and large-scale screening of diseases.

[0072] 4. The present invention demonstrates important value in the application of disease diagnosis. For diseases closely related to the metabolism of pipecolic acid and 6-oxopiperidinecarboxylic acid, such as pyridoxine-dependent epilepsy (PDE), the detection means provided by the present invention can accurately determine the content changes of these two substances in biological samples. Through the internal standard method of the present invention, using the internal standard substance 6-oxopiperidine-2-carboxylic acid-D3, which has specific deuterium-substituted hydrogen positions (2-position hydrogen substitution and 5-position hydrogen substitution of the piperidine ring), during the detection process, due to its highly similar structure and properties to the analyte, it can effectively calibrate the matrix effect and reduce the interference of complex components in the sample on the detection results. In actual operation, only by adding an appropriate amount of this internal standard solution to the sample to be tested and processing and analyzing it according to the established detection process, the effect of accurately quantifying the contents of pipecolic acid and 6-oxopiperidinecarboxylic acid can be achieved. Through the detection and analysis of a large number of clinical samples and combined with the scientific judgment criteria established by the present invention, it is possible to accurately judge whether the metabolic levels of these two substances in the patient's body are abnormal, thereby timely detecting potential patients with pyridoxine-dependent epilepsy. This not only helps in the early diagnosis and intervention of the disease, winning precious treatment time for patients, but also can monitor the patient's condition in real time during the treatment process, adjust the treatment plan according to the detection results, improve the pertinence and effectiveness of the treatment, and ultimately improve the patient's prognosis, playing a crucial role in the clinical diagnosis and treatment process of related diseases, and providing strong technical support for medical research and clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 : Synthetic route of the best internal standard substance 6-oxopiperidine-2-carboxylic acid-D3 of the present invention.

[0074] Figure 2 : Structural formula of the best internal standard substance 6-oxopiperidine-2-carboxylic acid-D3 of the present invention.

[0075] Figure 3 : of 6-oxopiperidine-2-carboxylic acid-D3 11H-NMR spectrum.

[0076] Figure 4 : of 6-oxopiperidine-2-carboxylic acid-D3 1 Partial enlarged view of the 1H-NMR spectrum. Specific implementation mode

[0077] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0078] In the present invention, the internal standard is D3-OXO-PIP: deuterated 6-oxopiperidinecarboxylic acid (i.e., 6-oxopiperidine-2-carboxylic acid-D3), and the internal standard is a stable isotope internal standard of 6-oxopiperidinecarboxylic acid (i.e., 6-oxopiperidine-2-carboxylic acid).

[0079] Example 1 Optimal synthesis method of the internal standard D3-OXO-PIP of the present invention

[0080] In the invention application with the publication number of CN118459394A and the invention title of "A Method for Preparing α-AASA and P6C", a method is proposed in which a macroporous resin is used to catalyze the AEA reaction, and then the macroporous resin is eluted to obtain an eluate (the eluate is not dried with nitrogen), which directly enters a specific chromatographic column for purification, and α-AASA and P6C with a relatively high purity level are obtained (specifically, the yield of α-AASA is 51%, the yield of P6C is 48%, and the total yield is 99%). However, in order to improve the chromatographic resolution of α-AASA and P6C, solve the problem of low chromatographic response, and the low purity and yield of the separated products, a derivatization operation is carried out in the method. During the derivatization process, the molecular structures of α-AASA and P6C are changed due to the introduction of new chemical groups. Since derivatization is mainly used for detection techniques, the changed molecular structures are no longer applicable to the purification process. At the same time, in the method, under the conditions of common chromatographic separation methods, α-AASA and P6C show obvious instability. α-AASA has a tendency to transform into P6C, and both of them will undergo degradation reactions. This instability causes a large loss of α-AASA during the reaction process, resulting in a seriously low yield. Although the yield of P6C is relatively slightly higher, the overall total yield is still at a low level. Therefore, in order to overcome these problems and further improve the preparation method, in subsequent research, steps such as fine optimization of the feeding ratio in the synthesis step, strict control of the elution conditions in the extraction step, and precise design of the mobile phase composition and gradient in the purification step are added, significantly increasing the difficulty and complexity of the preparation method of α-AASA and P6C.

[0081] α-AASA (α-aminoadipic semialdehyde) and P6C (δ1-Piperideine-6-carboxylate, that is, δ1-piperidine-6-carboxylate) play important roles in the metabolic process in vivo. In certain disease states, such as pyridoxine-dependent epilepsy (PDE), abnormal accumulation of α-AASA and P6C can lead to serious health problems. And 6-oxopiperidinecarboxylic acid has a certain association with α-AASA and P6C in the metabolic pathway. The deficiency of α-AASA dehydrogenase will lead to the accumulation of α-AASA and P6C. When α-AASA dehydrogenase is deficient, 6-hydroxyacrylic acid (6-OH-PIP) is oxidized to 6-oxopiperidinecarboxylic acid (6-oxohexanoate) by cytoplasmic enzymes. Therefore, accurate detection of 6-oxopiperidinecarboxylic acid is of great significance for studying the metabolism of α-AASA and P6C and the diagnosis of related diseases.

[0082] When detecting specific substances in body fluids such as blood and urine, the use of internal standards can significantly improve the accuracy of detection. Currently, there are many drawbacks in the existing synthesis methods of 6-oxopiperidinecarboxylic acid internal standards, such as low yield, complex operation, low purity, etc. Therefore, to solve the above problems, a new method for synthesizing the stable isotope internal standard of 6-oxopiperidinecarboxylic acid: 6-oxopiperidine-2-carboxylic acid-D3 is proposed in this embodiment. The specific synthesis route is as follows( Figure 1 )

[0083]

[0084] The synthesis route is as follows: In a sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in a mixed solution of 10 mL of methanol-D1 and 4 g of NaOD (40% in D2O), heat at 85 °C for 3 days. After cooling the solution to room temperature, concentrate the mixture, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, filter, and perform column chromatography after concentration under reduced pressure (eluent: V(methylene chloride) / V(methanol) = 10 / 1) to obtain 1.0 g of a white solid, which is 6-oxopiperidine-2-carboxylic acid-D3, and the yield reaches 83%. The purity and structure of the product are determined by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry. The structural formula of the 6-oxopiperidine-2-carboxylic acid-D3 is as Figure 2 shown, and its 1 1H-NMR spectrum is specifically as Figure 3 shown, Figure 4 which is a partial enlarged view of the 1 1H-NMR spectrum of 6-oxopiperidine-2-carboxylic acid-D3.

[0085] Therefore, it can be seen that the 6-oxopiperidine-2-carboxylic acid-D3 prepared by the best synthesis method of the present invention has a yield of up to 83% and a purity of up to 99%, and has the effect of significantly improving the accuracy and reliability of detection.

[0086] When detecting specific substances in body fluids such as blood and urine, the purity and yield of the internal standard are crucial for the accuracy of the detection results. A high-purity internal standard can reduce impurity interference and ensure the accuracy of the detection; while a higher yield can meet more detection needs and reduce the detection cost. The 6-oxopiperidine-2-carboxylic acid-D3 obtained by the synthesis method of the present invention has the characteristics of high yield and high purity, and can provide a stable and reliable internal standard for the detection process, thereby effectively improving the accuracy and reliability of the detection of 6-oxopiperidinecarboxylic acid and related substances such as α-AASA and P6C, and providing strong support for the diagnosis and research of related diseases.

[0087] Example 2 Screening of the Best Synthesis Method of the Internal Standard D3-OXO-PIP of the Present Invention

[0088] Since there are problems such as low yield, complex operation, and high cost in the synthesis method of the internal standard D3-OXO-PIP in the prior art, in order to further improve the efficiency, purity, and stability of D3-OXO-PIP synthesis in the prior art, in this embodiment, different synthesis methods were screened in order to prepare D3-OXO-PIP with high purity, high yield, simple operation, and low cost. The specific screening scheme is as follows:

[0089] 1. Scheme 1: Adopt the optimal synthesis method of the internal standard D3-OXO-PIP in Example 1, and the specific steps are as described in Example 1;

[0090] 2. Scheme 2: Adopt the synthesis method of the internal standard D3-OXO-PIP in the literature "Wempe MF, Kumar A, Kumar V, et al. Identification of a novel biomarker for pyridoxine-dependent epilepsy: Implications for newborn screening[J]. J Inherit Metab Dis, 2019, 42: 565-574. doi: 10.1002 / jimd.12059". The specific steps are as follows: The synthesis method of the internal standard d3-3,5,5-6-oxopiperic acid (D3-OXO-PIP) is: Dissolve deuterated α-aminoadipic acid (D3-AAA) (40 mg, 0.24 mmol) in 20% monodeuterated acetic acid (CH3COOD) / heavy water (D2O) (1.0 mL) and reflux at 108 °C for 3.5 hours, then evaporate the solvent under reduced pressure and dry under high vacuum for 1.0 hour. After that, add deuterated ethanol (CH3CH2OD; 10 mL), stir and filter twice. Combine the solutions obtained from the two filtrations, concentrate under reduced pressure and dry under high vacuum (4 hours), and 14 mg of deuterated 6-oxopiperidinecarboxylic acid with a purity greater than 97% is obtained by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry analysis;

[0091] The internal standard D3-OXO-PIP prepared by the synthesis method of Scheme 2 is specifically the internal standard with the hydrogen substitution at the 3-position and 5-position of the piperidine ring. During the synthesis process, first of all, there is a problem of poor selectivity in the deuteration reaction. Due to the existence of multiple active sites in the reaction system, it is extremely difficult to accurately achieve the substitution of hydrogen at the 3-position and 5-position. It is necessary to carry out extremely fine regulation of the reaction conditions. However, even so, it is still difficult to avoid the unnecessary deuteration or side reactions of hydrogen at other positions, which not only reduces the yield of the target product, but also makes the separation and purification of the product extremely difficult, increasing the complexity and cost of subsequent processing. The internal standard synthesized by the synthesis method of Example 1 of the present invention is specifically the internal standard with the hydrogen substitution at the 2-position and 5-position of the piperidine ring. In terms of reaction selectivity, this method has significant advantages. Its unique reaction design and condition optimization can accurately guide the deuteration reaction to occur only on the hydrogen at the 2-position and 5-position, effectively avoiding the unnecessary participation of hydrogen at other positions, thus ensuring a high generation efficiency of the target product.

[0092] At the same time, the yield of Scheme 2 is relatively low, only about 71%, which is quite different from 83% of Scheme 1. In addition, its operation is complex, involving relatively harsh conditions such as high-vacuum drying, which not only increases the equipment requirements and operation difficulty, but also raises the production cost. The high-vacuum drying process may cause partial loss of the product, further reducing the actual yield. Moreover, the harsh conditions require a relatively high technical level of the operators, and it is easy to have operation errors, affecting the stability of the product quality. In actual production applications, such a complex and harsh synthesis method may limit the feasibility of large-scale production, prolong the production cycle, and reduce the production efficiency.

[0093] Therefore, it can be seen that in terms of yield, the yield of Scheme 1 reaches 83%, which is significantly higher than that of Scheme 2. A high yield means that more target products can be obtained with the same raw material input, reducing the production cost and improving the production efficiency.

[0094] In terms of operation simplicity, the synthesis steps of Scheme 1 are relatively simple, without complex high-vacuum operations and multiple filtration steps, greatly reducing the operation time and labor cost. The operators do not need to have too high a technical level to complete the synthesis process, reducing the risk of operation errors and ensuring the stability of the product quality.

[0095] In terms of purity, although the purity of the product obtained by Method 2 is greater than 97%, it still fails to achieve the 99% purity effect of Method 1. Moreover, Method 1 has more advantages in terms of yield and operational simplicity, and the operational simplicity makes Method 1 easier to implement in practical applications. The synthesis steps of Method 1 are relatively concise, without the need for harsh conditions such as complex high-vacuum drying, reducing the operation time and labor costs. Operators do not need to have a high technical level to complete the synthesis process, reducing the risk of operation errors and ensuring the stability of the product quality. In addition, the simple operation process is also conducive to the realization of large-scale production, which can improve production efficiency and meet the market demand for high-purity internal standards.

[0096] In summary, only by adopting the optimal synthesis method of the internal standard D3-OXO-PIP in Example 1 of the present invention can an internal standard D3-OXO-PIP with high purity, high yield, simple operation and low cost be synthesized, which is the best choice of the present invention. This method provides a reliable internal standard synthesis route for detecting specific substances in body fluids such as blood and urine, helps to improve the accuracy and reliability of detection, can more accurately calibrate the matrix effect in the detection process, and thus shows higher accuracy, stability and repeatability in the detection of substances such as piperidine acid and 6-oxopiperidinecarboxylic acid, providing reliable guarantee for the diagnosis and research of related diseases.

[0097] Example 3 Optimization and improvement of the optimal synthesis method of the internal standard D3-OXO-PIP of the present invention

[0098] After screening out the optimal synthesis method of the internal standard D3-OXO-PIP, in order to further improve the yield, purity of D3-OXO-PIP and the stability and repeatability of the synthesis method, so as to reduce costs and improve production efficiency, in this example, different reaction conditions in the optimal synthesis method of the internal standard D3-OXO-PIP in Example 1 are optimized and screened. The specific scheme is as follows:

[0099] I. Optimization of reaction temperature

[0100] 1. Experimental design:

[0101] (1) Set different reaction temperature gradients: 80°C, 82°C, 85°C (control group, i.e., the temperature in Example 1), 88°C, 90°C;

[0102] (2) Take 5 sealed tubes. In each sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in a mixed solution of 10 mL of methanol-D1 and 4 g of NaOD (40% in D2O), and heat the 5 sealed tubes at different temperatures (80°C, 82°C, 85°C, 88°C, 90°C) for 3 days. The subsequent treatment steps are all the same as those in the optimal synthesis method in Example 1;

[0103] (3) Determine the purity and structure of the products in different sealed tubes by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry, and calculate the product yields at different temperatures;

[0104] 2. Result analysis:

[0105] Analyze the nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry of the reaction products at different temperatures. The specific results are shown in Table 1.

[0106] Table 1. Yields and purities of the internal standard D3-OXO-PIP at different temperatures

[0107] Reaction temperature Yield Purity 80℃ 75％ 92.6％ 82℃ 78％ 98.2％ 85℃ 83％ 99.5％ 88℃ 80％ 98.6％ 90℃ 72％ 85.7％

[0108] As can be seen from Table 1, when the temperature is in the range of 82 - 88 °C, the reaction rate is moderate, side reactions are few, and the best synthesis yields and purities are obtained. The purity of the internal standard D3-OXO-PIP can reach over 98%. And when the reaction temperature is 85 °C, the yields and purities of the internal standard D3-OXO-PIP are both optimal. This may be because within this temperature range, the energy supply of the reaction system is more appropriate. It can provide sufficient activation energy for the reaction to promote the effective collision and reaction between reactant molecules, and will not cause too many side reactions due to excessive energy. For the process of chemical bond breaking and formation involved in this reaction, especially the reaction steps related to deuterium substitution, this temperature range helps to maintain the selectivity and efficiency of the reaction, enabling the target product to be generated in a relatively high proportion, and ensuring the structural integrity and purity of the product.

[0109] When the reaction temperature is too low (80 °C and below), the energy of the reaction system is insufficient, the molecular movement rate slows down, and the effective collision frequency between reactants decreases significantly, resulting in an extremely slow reaction rate. A large amount of reactants fail to participate in the reaction sufficiently, thus making the yield of the final product at a relatively low level. At the same time, due to the extension of the reaction time, the probability of some unnecessary side reactions may increase. These side reactions may introduce impurities or cause partial degradation of the product, thereby affecting the purity of the product. When the reaction temperature is too high (90 °C and above), the excessive energy input makes the reaction system too active and easily triggers a series of complex side reactions. These side reactions may include the decomposition, isomerization, and unnecessary polymerization of reactants or products, etc. They not only consume a large amount of reactants, reducing the generation amount of the target product, but also produce a variety of impurities that are difficult to separate and remove, seriously affecting the purity of the product. In addition, too high a temperature may have an adverse effect on the chemical equilibrium of the reaction system, destroying the reaction equilibrium that is originally favorable for the generation of the target product, and further reducing the yield and purity of the product.

[0110] Therefore, it can be known that in this embodiment, 82-88°C is preferably used as the reaction temperature to ensure the high efficiency of the synthesis process and the high quality of the product. And when the preferred reaction temperature is 85°C, the yields and purities of the internal standard D3-OXO-PIP reach the best.

[0111] II. Optimization of reaction time

[0112] 1. Experimental design:

[0113] (1) Set different reaction times: 2 days, 2.5 days, 3 days (control group, i.e., the reaction time in Example 1), 3.5 days, 4 days;

[0114] (2) Take 5 sealed tubes. In each sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in a mixed solution of 10 mL of methanol-D1 and 4 g of NaOD (40% in D2O), and heat the 5 sealed tubes at 85°C for different times. The subsequent treatment steps are all the same as those in the best synthesis method in Example 1;

[0115] (3) Analytical method: Determine the purity and structure of the products in different sealed tubes by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry analysis, and calculate the product yields at different temperatures;

[0116] 2. Result analysis:

[0117] Analyze the nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry of the products under different reaction times. The specific results are shown in Table 2.

[0118] Table 2. Yields and purities of the internal standard D3-OXO-PIP under different reaction times

[0119] Reaction time Yield Purity 2 days 70％ 91.2％ 2.5 days 76％ 98.3％ 3 days 83％ 99.6％ 3.5 days 84％ 98.5％ 4 days 84％ 84.6％

[0120] As can be seen from Table 2, when the reaction time is 2.5 - 3.5 days, the best synthesis yield and purity are obtained, and the purity of the internal standard D3 - OXO - PIP can reach over 98%. And when the reaction time is 3 days, the yield and purity of the internal standard D3 - OXO - PIP are both optimal. This may be because in the initial stage of the reaction, such as a reaction time of 2 days, due to the too short reaction time, the number of effective collisions between reactant molecules is relatively small, and a large amount of reactants have not fully participated in the reaction to be converted into the target product, resulting in a yield of only 70%. At the same time, there may be more unreacted starting materials and a small amount of intermediate products generated due to incomplete reaction in the reaction system. The presence of these impurities leads to a relatively low level of product purity. As the reaction time is extended to 2.5 days, the reaction process is further promoted, and the reactants have more opportunities to undergo effective collisions and be converted into the target product, resulting in an increase in yield. At the same time, the reaction system gradually becomes stable, and some impurities further participate in the reaction or are removed through subsequent treatment steps during this process, thus significantly improving the product purity. When the reaction time reaches 3 days (control group), the reaction system reaches an ideal equilibrium state, and the reactants can be fully and efficiently converted into the target product D3 - OXO - PIP. At this time, the yield reaches 83%, and the purity of the product also reaches 99.6%. This indicates that at this time point, the selectivity and conversion rate of the reaction both reach the optimum, effectively reducing the occurrence of side reactions and the residue of impurities, ensuring the high quality of the product. However, when the reaction time continues to be extended to 3.5 days and 4 days, although the yield still slightly increases to 84% at 3.5 days, the purity begins to decline, and the purity drops to 84.6% at 4 days. This is because as the reaction time is overly extended, the reaction system may be affected by various adverse factors. On the one hand, too long a reaction time may cause side reactions such as degradation or isomerization of the generated target product, thereby reducing the product purity; on the other hand, under conditions such as long - term high temperature in the reaction system, some side reactions that were originally in a secondary position may be triggered. These side reactions not only consume the target product but also generate new impurities that are difficult to separate, further affecting the product purity and yield.

[0121] Therefore, it can be known that in this embodiment, it is preferred to use 2.5 - 3.5 days as the reaction time to ensure the high efficiency of the synthesis process and the high quality of the product. And when the preferred reaction time is 3 days, the yield and purity of the internal standard D3 - OXO - PIP are both optimal.

[0122] III. Necessity of adding methanol - D1 and NaOD for synergistic reaction

[0123] 1. Experimental design:

[0124] (1) Set up the control group: According to the method of Example 1, in a sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in a mixed solution of 10 mL of methanol-D1 and 4 g of NaOD (40% in D2O), and heat at 85 °C for 3 days.

[0125] (2) Set up experimental group 1: In a sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in 10 mL of methanol-D1, without adding NaOD, and heat at 85 °C for 3 days. The remaining conditions are the same as the optimal method in Example 1.

[0126] (3) Set up experimental group 2: In a sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in a mixed solution of 4 g of NaOD (40% in D2O), without adding methanol-D1, and heat at 85 °C for 3 days. The remaining conditions are the same as the optimal method in Example 1.

[0127] (4) Set up experimental group 3: In a sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in a mixed solution of 10 mL of methanol-D1 and 4 g of Na2CO3, and heat at 85 °C for 3 days. The remaining conditions are the same as the optimal method in Example 1.

[0128] (5) Set up experimental group 4: In a sealed tube, dissolve 1.2 g of 6-oxopiperidine-2-carboxylic acid in a mixed solution of 10 mL of methanol and 4 g of NaOD (40% in D2O), and heat at 85 °C for 3 days. The remaining conditions are the same as the optimal method in Example 1.

[0129] (6) Analysis method: Determine the purity and structure of the products in each experimental group by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry analysis. Compare the product yields of each experimental group with the control group, observe the progress of the reaction, and analyze the influence of different groups on the reaction.

[0130] 2. Result analysis:

[0131] Analyze the nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry of the products in different groups. The specific results are shown in Table 3.

[0132] Table 3. Yields and purities of the internal standard D3-OXO-PIP in different groups

[0133] Different groups Yield Purity Control group: Methanol-D1 + NaOD 83％ 99.6％ Experimental group 1: Only add Methanol-D1 18％ 22.5％ Experimental group 2: Only add NaOD 32％ 45.3％ <![CDATA[Experimental Group 3: Methanol-D1 + Na2CO3]]> 40％ 58.7％ Experimental group 4: Methanol + NaOD 64％ 85.2％

[0134] As can be seen from Table 3, only under the synergistic action of methanol-D1 and NaOD can the yield and purity of the internal standard D3-OXO-PIP reach the optimal level. When only methanol-D1 was added in Experimental Group 1, due to the lack of a suitable alkaline environment provided by NaOD, the reaction could not proceed effectively, resulting in extremely low yield and purity. This is because in the process of synthesizing 6-oxopiperidine-2-carboxylic acid-D3 from the molecular structure of 6-oxopiperidine-2-carboxylic acid, the alkaline strength and nature provided by NaOD are required to initiate key reaction steps, such as the activation and substitution processes of hydrogen atoms. Without NaOD, these reactions cannot proceed smoothly, and the reactants basically remain unchanged and cannot be converted into the target product. When only NaOD was added in Experimental Group 2, although there was an alkaline environment, due to the lack of the good dissolution and promotion effect of methanol-D1 on the reactants, the utilization rate of the reactants was low, and some reactants could not participate in the reaction fully, resulting in relatively low yield and purity. Methanol-D1 can increase the solubility of the reactants in the reaction system, enabling the reactant molecules to come into better contact and react, thereby improving the reaction efficiency. In Experimental Group 3, when Na2CO3 was used to replace NaOD, although it also provided an alkaline environment, the alkaline strength and catalytic effect of Na2CO3 were different from those of NaOD, and it could not promote the activation and transformation of the reactant molecules as precisely as NaOD, resulting in a slowdown in the reaction rate, incomplete reaction of some reactants, and possible adverse interactions with other components in the reaction system, interfering with the normal progress of the reaction, affecting the formation path and stability of the product, and thus significantly reducing the product yield and purity.

[0135] In Experimental Group 4, ordinary methanol was used to replace methanol-D1. The effect of ordinary methanol in promoting the reaction and increasing the solubility of the reactants was not as good as that of methanol-D1, resulting in a decrease in the reaction efficiency. The yield and purity were also lower than those of the control group, but there was still a certain degree of reaction compared with Experimental Groups 1 and 2.

[0136] In summary, only the synergistic action of methanol-D1 and NaOD can enable the reaction to obtain the target product with high purity at a high yield. Methanol-D1 and NaOD play indispensable roles respectively in the synthesis reaction of 6-oxopiperidine-2-carboxylic acid-D3, jointly promoting the efficient progress of the reaction and ensuring the high-quality output of the product.

[0137] IV. Optimization of the Dosages of Methanol-D1 and NaOD

[0138] 1. Experimental Design:

[0139] (1) Different dosages of methanol-D1 and NaOD were set, as shown in Table 4 specifically.

[0140] (2) Five sealed tubes were taken. In each sealed tube, 1.2 g of 6-oxopiperidine-2-carboxylic acid was dissolved in a mixed solution of methanol-D1 and NaOD (40% in D2O) with different dosage combinations and heated at 85 °C for 3 days. The subsequent treatment steps remained unchanged.

[0141] (3) Analytical method: The purity and structure of the products in different sealed tubes were determined by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry analysis, and the product yields at different temperatures were calculated.

[0142] 2. Result analysis:

[0143] The nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry of the products in different groups were analyzed, and the specific results are shown in Table 4.

[0144] Table 4. Yields and purities of the internal standard D3-OXO-PIP in different groups

[0145] Dosage of Methanol-D1 Dosage of NaOD Yield Purity 5 mL 3.5g 66％ 68.5％ 5 mL 3.8g 70％ 86.7％ 5 mL 4g 71％ 88.2％ 5 mL 4.2g 73％ 85.3％ 5 mL 4.5g 69％ 70.3％ 8 mL 3.5g 75％ 69.8％ 8 mL 3.8g 79％ 98.4％ 8 mL 4g 81％ 99.2％ 8 mL 4.2g 80％ 98.3％ 8 mL 4.5g 74％ 72.6％ 10 mL 3.5g 76％ 75.3％ 10 mL 3.8g 80％ 98.3％ 10 mL 4g 84％ 99.8％ 10 mL 4.2g 81％ 98.5％ 10 mL 4.5g 75％ 73.8％ 12 mL 3.5g 74％ 71.5％ 12 mL 3.8g 78％ 98.5％ 12 mL 4g 82％ 99.0％ 12 mL 4.2g 81％ 98.6％ 12 mL 4.5g 76％ 72.8％ 15 mL 3.5g 64％ 67.4％ 15 mL 3.8g 69％ 86.2％ 15 mL 4g 72％ 88.6％ 15 mL 4.2g 71％ 85.4％ 15 mL 4.5g 67％ 68.2％

[0146] It can be seen from Table 4 that when the dosage of methanol-D1 is in the range of 8 - 12 mL and the dosage of NaOD is in the range of 3.8 - 4.2 g, the yields and purities of the products are at relatively high levels. And the best effect is achieved when methanol-D1 is 10 mL and NaOD is 4 g.

[0147] This may be because in this dosage combination, the reaction system reaches the optimal equilibrium state. As a reaction auxiliary solvent, when the dosage of methanol-D1 is 10 mL, it can provide a suitable dissolution environment for the reactants, ensuring that the reactant molecules are fully dispersed in the reaction system, enabling sufficient contact opportunities between the reactants and NaOD, and promoting the progress of the reaction. At the same time, 4 g of NaOD can provide just the right alkaline strength, neither triggering excessive side reactions due to overly strong alkalinity nor failing to provide enough activation energy for the reaction, enabling the hydrogen atoms in the molecular structure of 6-oxopiperidine-2-carboxylic acid to be efficiently activated and undergo substitution reactions, converting into the target product 6-oxopiperidine-2-carboxylic acid-D3, thus achieving the synthesis of products with high yield and high purity. When the dosage of methanol-D1 or NaOD deviates from this optimal range, the balance of the reaction system is disrupted, which may lead to problems such as reduced utilization rate of reactants, increased side reactions, or incomplete reactions, thereby affecting the yield and purity of the products. For example, when the dosage of NaOD is 3.5 g, due to relatively insufficient alkali amount, the alkaline strength of the reaction system fails to reach the optimal catalytic effect, resulting in a limited reaction rate between reactant molecules, and some reactants cannot be fully converted into the target product, thus significantly reducing the yield. At the same time, the relatively low alkaline environment is also not conducive to the selectivity of the reaction, and may trigger some side reactions, resulting in a relatively low level of product purity. As the dosage of NaOD increases, the alkalinity of the reaction system increases, and more reactants can participate in the reaction under the catalysis of suitable alkalinity, resulting in an increase in yield, and the selectivity of the reaction also improves to a certain extent, the generation of impurities is inhibited, and the product purity is significantly improved. When the dosage of NaOD reaches 4 g (control group), the reaction system reaches the ideal alkaline condition and the conversion equilibrium state of reactants, and the reactants can be efficiently and accurately converted into the target product D3-OXO-PIP. At this time, both the yield and purity reach the best, fully indicating that the activity and selectivity of the reaction reach the optimal combination at this dosage, effectively ensuring the high-quality output of the product. When the dosage of NaOD is further increased (4.5 g), the yield and purity also decrease significantly. This is because too high a dosage of NaOD will make the alkalinity of the reaction system too strong, triggering a series of adverse side reactions. The overly strong alkaline environment may cause excessive reactions of reactants or the already formed products, such as decomposition, isomerization, or unnecessary acid-base reactions with the excessive alkali, thus consuming a large amount of reactants and target products, reducing the yield. At the same time, these side reactions will also generate a variety of impurities that are difficult to separate and remove, seriously affecting the product purity, making the product quality unable to meet the high standards required for practical applications.

[0148] Therefore, it can be known that in this embodiment, it is preferable that the dosage of methanol-D1 is in the range of 8 - 12 mL and the dosage of NaOD is in the range of 3.5 - 4.5 g. At this time, the purity of the product D3-OXO-PIP can reach over 98%. At the same time, the effect is the best when methanol-D1 is 10 mL and NaOD is 4 g.

[0149] V. Optimization of Eluent Ratio

[0150] 1. Experimental Design:

[0151] (1) Adjust the ratio of V(methylene chloride):V(methanol) in the eluent and set different ratio gradients: 8:1, 9:1, 10:1 (control group, i.e., the volume ratio of methylene chloride and methanol in Example 1), 11:1, 12:1.

[0152] (2) Take 5 sealed tubes. After the reaction in the sealed tubes, process them to the column chromatography stage according to the conventional steps, and use eluents with different ratios for column chromatography in each sealed tube.

[0153] (3) Analytical method: Determine the purity and structure of the products in different sealed tubes by nuclear magnetic resonance and liquid chromatography - tandem mass spectrometry analysis, and determine the optimal eluent ratio through the analysis of the products.

[0154] 2. Result Analysis

[0155] Analyze the nuclear magnetic resonance and liquid chromatography - tandem mass spectrometry of the products in different groups. The specific results are shown in Table 5.

[0156] Table 5. Yield and Purity of Internal Standard D3 - OXO - PIP in Different Groups

[0157]

[0158]

[0159] As can be seen from Table 5, when the ratio of V(methylene chloride):V(methanol) in the eluent is 9:1 - 11:1, the best synthesis yield and purity can be achieved, and the purity of the internal standard D3-OXO-PIP can reach over 98%. Moreover, when the ratio of V(methylene chloride):V(methanol) in the eluent is 10:1, the yield and purity of the internal standard D3-OXO-PIP reach the best. This is because during the column chromatography process, the ratio of the eluent plays a crucial role in the separation effect of the target product and impurities. Different ratios of methylene chloride and methanol will affect the polarity and elution ability of the eluent. When the ratio is 10:1, the polarity of the eluent is moderate, which can not only ensure sufficient elution ability for the target product D3-OXO-PIP to effectively elute it from the stationary phase, but also avoid over-eluting impurities, thus achieving good separation of the target product and impurities, with the yield reaching 83% and the purity as high as 99.5%. When the ratio of V(methylene chloride):V(methanol) in the eluent is 8:1, the proportion of methylene chloride is relatively low, the polarity of the eluent is strong, and the elution ability is weak, resulting in some of the target product being difficult to be completely eluted from the stationary phase, with the yield only being 76%. At the same time, some impurities with similar polarities may be eluted together with the target product, affecting the product purity and keeping it at a relatively low level. When the ratio is 12:1, the proportion of methylene chloride is too high, the polarity of the eluent is too weak. Although it may enhance the elution ability for some impurities, it is also easy to bring out some impurities that should not be eluted, and at the same time, the retention time of the target product in the column may be too long, resulting in partial decomposition or adsorption loss, etc., and thus the yield and purity are also significantly reduced.

[0160] When the ratio between the two is within 9:1 - 11:1, it can better balance the elution ability and separation effect, ensuring the efficient recovery and high purity of the target product. Therefore, in this example, the ratio of V(methylene chloride):V(methanol) in the eluent is preferably 9:1 - 11:1. And when the ratio between the two is 10:1, the yield and purity of the internal standard D3-OXO-PIP reach the best, providing a reliable condition guarantee for the high-quality preparation of the subsequent internal standard.

[0161] Example 4 Influence of Different Detection Methods on Detection Results

[0162] Pyridoxine-dependent epilepsy (PDE) is a rare genetic metabolic disorder mainly caused by gene mutations that lead to an increased dependence of the body on vitamin B6 (pyridoxine). Patients with PDE experience frequent seizures in the neonatal period or early infancy and usually do not respond well to conventional anti-epileptic drug treatments. Without timely and effective treatment, it may cause severe neurological damage, such as intellectual developmental delay, movement disorders, speech disorders, etc., seriously affecting the quality of life and prognosis of patients. Therefore, high accuracy and sensitivity are required in the screening diagnosis and intervention treatment of PDE. This is because the early symptoms of PDE may not be obvious and the disease progression is relatively complex. If diagnosis and treatment cannot be carried out promptly and accurately, serious consequences may occur.

[0163] In previous studies, the inventors used a derivatization detection method that required precise quantitative determination of five substances: piperidine-6-carboxylic acid (P6C), ɑ-aminoadipic semialdehyde (ɑ-AASA), ɑ-aminoadipic acid (AAA), pipecolic acid (PA), and 6-oxopiperidinecarboxylic acid (6-OXO-PIP). Based on this, the preliminary screening and diagnostic evaluation of PDE could be achieved. However, this method has significant drawbacks. Its operation process is extremely complex, involving the detection of multiple substances and having extremely high requirements for the control of experimental conditions. This undoubtedly increases the detection cost and time cost, and also increases the probability of detection errors.

[0164] In contrast, the present invention innovatively uses the internal standard method for detection. The main function of the derivatization solution is to chemically modify the target substance, making it easier to be detected and identified in the subsequent detection process, thereby improving the sensitivity and accuracy of the detection. In the present invention, 6-oxopiperidine-2-carboxylic acid-D3 is directly used as the internal standard without going through complex derivatization steps. By virtue of the high consistency of the physical and chemical properties between the internal standard and the analyte, the matrix effect can be accurately calibrated and the influence of interference factors on the detection result can be reduced. Specifically, as the internal standard, 6-oxopiperidine-2-carboxylic acid-D3 has deuterium-substituted hydrogen positions (the 2-position and 5-position hydrogen substitution of the piperidine ring), which makes it have similar retention behavior and ionization efficiency to the analyte during chromatographic analysis. During the detection process, the internal standard can accurately track the elution process of the analyte, effectively reducing problems such as peak tailing and peak area changes caused by the complex matrix components. By using the internal standard method, the present invention not only simplifies the detection process but also significantly improves the accuracy and repeatability of the detection, enabling accurate quantitative detection of piperidine acid and 6-oxopiperidine carboxylic acid in complex biological samples. During the actual detection process, only the combined detection of two key substances, piperidine acid (PA) and 6-oxopiperidine carboxylic acid, needs to be carried out. By accurately measuring their content levels in biological samples, the PDE disease can be diagnosed efficiently and accurately, and the disease condition can be monitored in real time. More importantly, this method can significantly improve the accuracy and sensitivity of PDE disease diagnosis, providing strong support for clinical diagnosis. This may be due to the following reasons: on the one hand, as the internal standard, 6-oxopiperidine-2-carboxylic acid-D3 has specific deuterium-substituted hydrogen positions, making it highly similar in structure and properties to the analyte piperidine acid and 6-oxopiperidine carboxylic acid. It can effectively calibrate the matrix effect during the detection process and reduce the influence of interference factors on the detection result, thereby improving the accuracy and sensitivity of the detection. On the other hand, focusing on the combined detection of two key biomarkers, piperidine acid and 6-oxopiperidine carboxylic acid, reduces the complexity and workload of the detection, and also improves the pertinence of the detection. Research shows that these two substances play an important indicative role in the occurrence and development of PDE disease. By accurately measuring their content levels, the disease state can be more directly reflected, providing more valuable information for the diagnosis and treatment of the disease.

[0165] Therefore, in order to verify that the detection method of the present invention (internal standard method: using 6-oxopiperidine-2-carboxylic acid-D3 prepared by the optimal synthesis method in Example 1 of the present invention as the internal standard) has the best detection and quantification effect on piperidine acid and 6-oxopiperidine carboxylic acid, in this example, a comparative analysis of different detection methods was carried out as follows:

[0166] I. Detection conditions:

[0167] 1. Chromatographic conditions: Mobile phase A: water; Mobile phase B: methanol and acetonitrile, with the volume ratio of methanol to acetonitrile being 1:9. For the conditions not mentioned otherwise, they are the same as those described in the best conditions of the invention application with the application publication number CN118459394A and the invention title "A method for preparing α - AASA and P6C", but the derivatization step is not carried out.

[0168] The elution gradient is shown in Table 6.

[0169] Table 6. Elution gradient

[0170] Time (min) Flow rate (mL / min) A％ B％ 0 0.4 90 10 1 0.4 90 10 1.1 0.4 65 35 1.5 0.4 55 45 1.7 0.4 30 70 2.5 0.4 25 75 2.6 0.4 0 100 3.6 0.4 0 100 4 0.4 90 10 5 0.4 90 10

[0171] 2. Sample treatment:

[0172] (1) Take 100 μL of the sample to be tested in a 1.5 mL centrifuge tube, add 50 μL of the D3 - OXO - PIP internal standard working solution and mix well. Then add 200 μL of the precipitant, vortex - mix for 1 min, centrifuge at 1000 g for 5 min at 4 °C, separate the supernatant, and discard the protein precipitate. In the present invention, the precipitant includes any one of methanol, acetonitrile, acetone, and ethanol, specifically methanol.

[0173] (2) Take the supernatant from step (1), dry it under nitrogen, and dissolve it with the reconstitution solution (methanol: water = 1:1) to obtain the sample extract.

[0174] (3) Perform liquid chromatography analysis on the sample extract, with an injection volume of 20 μL.

[0175] II. Comparison results of detections using different detection methods

[0176] Select PDE - positive blood samples and PDE - negative blood samples that have been clinically diagnosed and have clear diagnostic results respectively. For each sample type, the following three different detection methods are used for analysis: (1) Use the internal standard method (quantify pipecolic acid and 6 - oxopiperidinecarboxylic acid); (2) Use the derivatization method for detection (quantify pipecolic acid and 6 - oxopiperidinecarboxylic acid); (3) Use the derivatization method for detection (quantify piperidine - 6 - carboxylic acid, α - aminocaproic semialdehyde, α - aminocaproic acid, pipecolic acid, 6 - oxopiperidinecarboxylic acid). Each method is repeated three times, the peak areas of different substances are detected respectively, and the CV value is calculated to evaluate the repeatability and precision of the detection method. At the same time, the accuracy of different detection methods is judged according to the known samples to ensure the reliability and stability of the experimental data. The specific results are shown in Tables 7 - 9.

[0177] Table 7. Detection results of the internal standard method

[0178]

[0179]

[0180] Table 8. Detection Results of Derivatization Method (Quantitative Analysis of Piperidinecarboxylic Acid and 6-Oxopiperidinecarboxylic Acid)

[0181]

[0182] Table 9. Detection Results of Derivatization Method (Quantitative Analysis of Piperidine-6-carboxylic Acid, α-Aminoadipic Semialdehyde, α-Aminoadipic Acid, Piperidinecarboxylic Acid, and 6-Oxopiperidinecarboxylic Acid)

[0183]

[0184]

[0185] In the present invention, the method for judging positive and negative samples is as follows: In the internal standard method of the present invention, an internal standard substance with a known concentration (such as 6-oxopiperidine-2-carboxylic acid-D3) is detected together with the analyte (piperidinecarboxylic acid and 6-oxopiperidinecarboxylic acid). By establishing a standard curve, the peak area ratio of the analyte to the internal standard substance is substituted into the curve equation to calculate the concentration. If it exceeds the upper limit of the normal reference range, it is judged as positive; if it is within the range, it is judged as negative. Specifically, when the peak area (PA) ≥ 1000, the sample is positive; when the peak area (PA) < 1000, the sample is negative; when the peak area (6-OXO-PIP) ≥ 1300, the sample is positive; when the peak area (6-OXO-PIP) < 1300, the sample is negative. In the derivatization method, the standard product is first derivatized and a standard curve is established. The judgment is based on the absolute peak area of the target substance or a simple peak area ratio. When the peak area or its ratio of PA and 6-oxopiperidinecarboxylic acid in the sample is similar to the characteristic peak area or ratio of the positive sample, it is judged as positive; when it is similar to the characteristics of the negative sample, it is judged as negative. Specifically, when the peak area (P6C) ≥ 850000, the sample is positive; when the peak area (P6C) < 850000, the sample is negative; when the peak area (α-AASA) ≥ 35000, the sample is positive; when the peak area (α-AASA) < 35000, the sample is negative; when the peak area (AAA) ≥ 65000, the sample is positive; when the peak area (AAA) < 65000, the sample is negative; when the peak area (PA) ≥ 1000, the sample is positive; when the peak area (PA) < 1000, the sample is negative; when the peak area (6-OXO-PIP) ≥ 1300, the sample is positive; when the peak area (6-OXO-PIP) < 1300, the sample is negative.

[0186] As can be seen from the results in Tables 7 - 9, the internal standard method of the present invention shows excellent performance in terms of detection accuracy, sensitivity, repeatability, and precision. The CV values are all less than 10%, reaching the best level. Moreover, during multiple repeated detections, the stability and accuracy of the positive and negative judgment results of the samples are extremely high. This indicates that the best internal standard method of the present invention can effectively reduce experimental errors and ensure the accuracy and consistency of each detection result. This benefits from the unique advantages of the 6 - oxopiperidine - 2 - carboxylic acid - D3 internal standard. Its specific deuterium - substituted hydrogen position makes it highly similar in structure and properties to the piperidine acid and 6 - oxopiperidine carboxylic acid to be detected, enabling it to efficiently calibrate the matrix effect during the detection process, effectively reducing the influence of interference factors on the detection result, and thus accurately distinguishing PDE positive and negative samples.

[0187] In contrast, when only quantitatively detecting piperidine acid and 6 - oxopiperidine carboxylic acid using the derivatization method, although the peak area data of these two substances can be obtained, the judgment of positive or negative results of the samples is extremely unstable, with a large number of misjudgments occurring. This is mainly because detecting only these two substances cannot comprehensively reflect the metabolic state of the organism and is easily interfered by other factors, resulting in incorrect judgments. In addition, the conditions of the derivatization reaction are difficult to precisely control. Minor changes in temperature and time may lead to differences in the reaction results, thereby affecting the accuracy of quantification. At the same time, the purity and stability of the derivatization reagent are difficult to guarantee, which may introduce impurities to interfere with the detection, further reducing the reliability of the detection result.

[0188] When quantitatively detecting the five substances of 6 - hydroxypiperidine acid, α - aminoadipic semialdehyde, α - aminoadipic acid, piperidine acid, and 6 - oxopiperidine carboxylic acid using the derivatization method, although positive and negative samples can be accurately judged, this method is complex to operate. It requires separate treatment and analysis of the five substances, which is time - consuming and prone to introducing errors. This undoubtedly increases the difficulty and cost of detection and has great limitations in actual clinical applications. At the same time, its CV values are also higher than those of the internal standard method of the present invention.

[0189] Therefore, it can be known that the internal standard method of the present invention has obvious advantages in the detection of PDE diseases. It not only has good detection repeatability and precision, can provide reliable data support for clinical diagnosis, but also has high accuracy, can accurately judge positive and negative samples, and greatly improves the efficiency and reliability of PDE disease diagnosis.

[0190] Example 5 Influence of internal standards synthesized by different methods on detection results

[0191] In the detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, a suitable internal standard is crucial for improving the precision and accuracy of the detection method. For the internal standard 6-oxopiperidine-2-carboxylic acid-D3 of the present invention, differences in its synthesis route will significantly change the characteristics of the internal standard, resulting in significant differences in the detection results for internal standards synthesized by different methods.

[0192] The 6-oxopiperidine-2-carboxylic acid-D3 synthesized by the optimal synthesis method of the present invention has deuterated hydrogen substitution positions at the 2-position hydrogen and 5-position hydrogen of the piperidine ring, ensuring the stability and specificity of the internal standard. However, for the internal standard obtained by the synthesis method provided in Scheme 2 of Example 2, the deuterated hydrogen substitution positions are at the 3-position hydrogen and 5-position hydrogen of the piperidine ring, which are different from those of the present invention, resulting in a reduced matching degree between the internal standard and the analyte in terms of structure and properties.

[0193] To verify the influence of internal standards synthesized by different methods on the detection results, the following experiments were conducted:

[0194] Influence of Internal Standards Synthesized by Different Methods on Detection Results

[0195] 1. Preparation of two different internal standards:

[0196] The specific preparation processes and results are as described in Example 2. Internal standard 1 (D3-OXO-PIP-1 prepared by the optimal synthesis method in Example 1) and internal standard 2 (D3-OXO-PIP-2 synthesized in Scheme 2 of Example 2) were respectively prepared.

[0197] 2. Influence of different internal standards on sample detection:

[0198] Sample quantitative detection experiments were respectively carried out using internal standard 1 and internal standard 2. During the experiment, a series of representative samples were selected, including standard solution 1 (10 ng / mL) and standard solution 2 (100 ng / mL) with different concentration gradients, as well as actual PDE biological negative samples, to ensure that the experimental results can comprehensively reflect the performance of different internal standards in various sample types and concentration ranges. For each sample, the detection method was the same, as specifically described in Example 4.

[0199] Table 10. Results of the Influence of Different Internal Standards on Sample Detection

[0200]

[0201]

[0202] Note: The coefficient of variation (CV) is a statistic that measures the degree of data dispersion, representing the ratio of the standard deviation to the mean, which reflects the relative fluctuation of the data. Its calculation formula is CV = SD / M, where SD is the standard deviation, representing the degree of dispersion of a set of data; M is the mean, reflecting the central tendency of the data. In this experiment, by conducting multiple tests on each internal standard on different quality control samples, the mean and standard deviation were calculated, and then the coefficient of variation was obtained. The smaller the coefficient of variation, the smaller the degree of data dispersion, and the better the stability and repeatability of the test results.

[0203] As can be seen from Table 10, when using internal standard 1 prepared by the optimal synthesis method of the present invention, more accurate and stable peak areas can be obtained in the quantitative detection of pipecolic acid and 6-oxopiperidinecarboxylic acid. During multiple repeated detections, when using internal standard 1, the measured CV values are all less than 10%, and are significantly lower than the CV values when using internal standard 2. This indicates that internal standard 1 can more effectively correct errors in the detection process, such as matrix effects, instrument fluctuations, etc., thus significantly improving the precision and accuracy of the test results. This may be because the deuterium-hydrogen substitution positions of internal standard 1 (hydrogen substitution at the 2-position and 5-position of the piperidine ring) make its chemical structure more similar to the analytes pipecolic acid and 6-oxopiperidinecarboxylic acid. During the detection process, internal standard 1 can better simulate the behavior of the analytes, and has higher consistency with the analytes in terms of retention time on the chromatographic column, ionization efficiency, etc. When there is a matrix effect, the interaction mode between internal standard 1 and matrix components is closer to that of the analytes, so that it can more accurately offset the influence of the matrix on the test results. During the instrumental analysis process, due to its structural similarity, internal standard 1 can maintain a more stable proportional relationship with the analytes in terms of instrument signal response, reducing the detection errors caused by instrument fluctuations.

[0204] In contrast, the positions of deuterium-hydrogen substitution in internal standard 2 (hydrogen substitution at the 3-position and 5-position of the piperidine ring) have relatively large structural differences from the analyte. In actual detection, its elution behavior on the chromatographic column may deviate from that of the analyte to a certain extent, resulting in peak broadening or unstable retention time, which in turn affects the accurate measurement of peak area. During the ionization process, due to the structural differences, the correlation between its ionization efficiency and the analyte is weak, causing the ratio of the response signals of internal standard 2 to the analyte to fluctuate greatly in different samples or detection batches, unable to effectively correct the detection error, and reducing the precision and accuracy of the detection results. At the same time, during the synthesis process, the substitution of hydrogen at the 3-position is more difficult because the chemical environment where the 3-position hydrogen atom is located is more complex than that of the 2-position hydrogen atom, and the distribution of the surrounding electron cloud and steric hindrance situation have a greater hindrance to the deuteration reaction. When carrying out the deuteration reaction, more stringent reaction conditions and more precise reaction control are required, which not only increases the cost and complexity of synthesis, but also easily leads to the occurrence of side reactions, reducing the purity of the product and being unfavorable for large-scale industrial production. In addition, due to the difficulty in precisely controlling the degree and selectivity of hydrogen substitution at the 3-position, the differences between batches of the synthesized internal standard 2 are relatively large, further affecting its stability and reliability during the detection process, unable to meet the requirements of high-precision detection, thus limiting its effectiveness in practical applications.

[0205] Therefore, it can be seen that in the detection of pipecolic acid and 6-oxopiperidinecarboxylic acid, it is crucial to select a suitable synthesis method for the internal standard. The internal standard 1 prepared by the optimal synthesis method of the present invention has obvious advantages, can provide a more reliable quantitative basis for related detections, helps to improve the overall performance of the detection method, and has important application value in fields such as disease diagnosis and biological sample analysis. Such an internal standard should be preferentially selected for such detection work to ensure the accuracy and reliability of the detection results.

[0206] The above-described embodiments have elaborated on the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kit for the combined detection of pipecolic acid and 6-oxonipecotic acid, characterized in that: The invention comprises an internal standard solution, wherein the internal standard solution comprises an internal standard substance 6-oxopiperidine-2-carboxylic acid-D3, wherein the substitution position of deuterated hydrogen in the internal standard substance comprises one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th and 5th positions of the piperidine ring, or hydrogen substitution at the carbon atom connected with a formic acid group.

2. The kit according to claim 1, characterized in that In the internal standard, the substitution positions of deuterated hydrogen include hydrogen substitution at the 2-position and hydrogen substitution at the 5-position of the piperidine ring; the structural formula of the 6-oxopiperidine-2-carboxylic acid-D3 is as follows:

3. A combined detection method for pipecolic acid and 6-oxonipecotic acid, characterized in that: The detection is performed using the kit as described in any one of claims 1 to 2.

4. The joint detection method according to claim 3, characterized in that: The following steps are involved: (1) Add an internal standard solution to the sample to be tested, then add a precipitant, mix well, centrifuge, separate the supernatant, and discard the protein precipitate; (2) taking the supernatant in step (1), drying it with nitrogen gas, adding the reconstitution solution to dissolve it, and obtaining a sample extract; (3) Perform liquid chromatography on the sample extract.

5. The joint detection method according to claim 4, characterized in that: In step (1), the precipitant includes any one or more combinations of methanol, acetonitrile, acetone and ethanol.

6. The joint detection method according to claim 5, characterized in that: In step (1), the volume ratio of the internal standard solution to the precipitant is 1:(2-5).

7. A composition for preparing a reagent for improving the accuracy and sensitivity of the combined detection of pipecolic acid and 6-oxonipecotic acid, characterized in that: The composition comprises an internal standard and a precipitant; the internal standard comprises 6-oxopiperidine-2-carboxylic acid-D3, and the precipitant comprises methanol.

8. The use according to claim 7, characterized in that In the internal standard, the substitution position of deuterated hydrogen includes one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th, 5th position of the piperidine ring, or hydrogen substitution at the carbon atom to which the formic acid group is connected.

9. The use according to claim 8, characterized in that In the internal standard, the substitution positions of deuterated hydrogen include hydrogen substitution at the 2-position and hydrogen substitution at the 5-position of the piperidine ring; the structural formula of the 6-oxopiperidine-2-carboxylic acid-D3 is as follows:

10. A use of 6-oxopiperoidine-2-carboxylic acid-D3 for preparing an internal standard solution for improving the accuracy and sensitivity of combined detection of pipecolic acid and 6-oxopiperoidine carboxylic acid, wherein the substitution position of deuterated hydrogen in the 6-oxopiperoidine-2-carboxylic acid-D3 includes one or more of the following: hydrogen substitution at the 2nd, 3rd, 4th, or 5th position of the piperidine ring, or hydrogen substitution at the carbon atom to which the formic acid group is connected.

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