Kit for detecting vitamin B6 and metabolite thereof and use method of kit

Abstract

The invention provides a kit for detecting vitamin B6 and metabolites thereof and a use method of the kit, and relates to the field of metabolite detection. The kit comprises an internal standard prepared from malic acid, a calibrator solution and a TCA protein precipitant. The use method of the kit comprises the following steps: adding a blood sample into an internal standard solution, then adding TCA, carrying out vortex centrifugation, taking a supernatant, analyzing the supernatant by using an LC-MS / MS technology, and then detecting to obtain the concentrations of vitamin B6 and metabolites (PLP, PL, PA, PN and PNP) thereof in blood plasma or red blood cells. The malic acid is used for inhibiting degradation of PLP, the accuracy of the detection result is guaranteed, the kit can detect various forms of vitamin B6 at the same time, the detection time is short, and the clinical prospect is huge.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of metabolite detection, in particular to a kit for detecting vitamin B6 and its metabolites and a method of use thereof. BACKGROUND

[0002] Vitamin B6 is a general term for a class of compounds, specifically pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their phosphorylated derivatives. Vitamin B6 is associated with many physiological diseases, such as cardiovascular disease, cancer development, oxidative stress, chromosomal instability and inflammation. Human bodies do not have the ability to synthesize vitamin B6, but have a sophisticated mechanism for converting various forms of vitamin B6 into each other, which is important for the absorption, transport and bioavailability of vitamin B6, and is embodied in the diversified use of different forms of vitamin B6. Vitamin B6 exists in various forms in the body, but is ultimately metabolized into pyridoxic acid (PA) and excreted from the body, and the level of PA can reflect the metabolic state and nutritional status of vitamin B6 in the body. In addition, vitamin B6 plays an important role in human metabolism, among which pyridoxal phosphate (PLP) is the active form of vitamin B6, about 4% of enzymes in the human body rely on PLP to function, so PLP is necessary for many enzymatic reactions. Deficiency or abnormally high levels of PLP have a negative impact, and in many clinical situations, measuring the concentration of PLP and related metabolites is necessary for diagnosis and monitoring.

[0003] However, current patents mainly provide methods for detecting a single form of vitamin B6 (such as pyridoxine or PLP) and its impurities in vitamin B6 supplements, such as the patents "Method for detecting vitamin B-6 and related substances thereof" (Application No. 202111650325.0) and "Method for detecting vitamin B6 injection and its impurities using HPLC" (Application No. 202311762715.6). These methods have few detection indicators, complex sample pretreatment, and long detection times. Although there are some methods for detecting the content of vitamin B6 in human samples, these methods require a long detection time and can only detect a specific form of vitamin B6, such as "Kit for detecting vitamin B6 and its detection method" (Application No. 202310205652.8) and the article "Simultaneous measurement of whole blood vitamin B1 and vitamin B6 using LC-ESI-MS / MS" [1]. In summary, there is no method that can simultaneously detect multiple forms of vitamin B6 and its metabolites. Therefore, it is necessary to develop new technologies to simplify, accurately, and reproducibly detect the content of different forms of vitamin B6 in the human body at the same time, and to develop a stable detection reagent to ensure the long-term stability of each substance in the reagent, such as preventing PLP from converting to PL and PA, thereby improving the reproducibility, convenience, and accuracy of detection. SUMMARY

[0004] Vitamin B6 is an important vitamin in the metabolic process of the human body, and its level affects a series of metabolic processes. Phosphopyridoxal, for example, is a key coenzyme known to participate in various enzymatic reactions. However, it is difficult to stably and accurately quantify vitamin B6 and related metabolites in blood samples, which is mainly limited by the instability of vitamin B6 and related metabolites in the sample. Metabolites can convert to each other, such as the standard solution of PLP rapidly converts to PL and PA during storage. The degradation rate of PLP in pure water solution stored at -80℃ for one month exceeds 50%. However, current literature and patents do not provide technical solutions for inhibiting PLP degradation and simultaneously detecting vitamin B6 and related metabolites.

[0005] To solve the above problems, the application not only provides a solution to the problem of converting PLP into PL and PA, i.e., a 1% malic acid (MA) solution can significantly increase the stability of PLP, which may be because MA acts as a chelating agent and a pH regulator; but also develops a method based on LC-MS / MS technology for stably detecting the content of vitamin B6 and its metabolites (PLP, PL, PA, PN and PNP) in blood samples (plasma and red blood cells), specifically, using a 25% TCA solution to precipitate the heteroprotein in the sample, then taking the supernatant, and using the LC-MS / MS technology to accurately and quickly detect PLP, PL, PA, PN and PNP in plasma and red blood cells, which will help to clarify the role of vitamin B6 and related metabolites in the occurrence and development of diseases, and has great clinical significance.

[0006] To achieve the above object, the technical scheme adopted by the application is as follows:

[0007] On the one hand, the application provides the use of organic acids for preparing reagents for stabilizing pyridoxal phosphate and / or detecting vitamin B6 and its metabolites.

[0008] In the preliminary experiments of the application, it was found that pyridoxal phosphate (PLP) would be converted into PL and PA, and through literature review, it was speculated that the reasons for the degradation of PLP might be that PLP was catalytically degraded by metal ions in the aqueous solution, or that the pH value could not stabilize PLP, and the presence of microorganisms and the occurrence of oxidation reaction, etc.; and when using LC-MS / MS technology to detect PLP, its standard and calibration solution and internal standard are inevitably used, and if the content of PLP in the standard and / or calibration solution and / or internal standard solution is not constant, it will lead to inaccurate detection results and poor experimental repeatability, therefore, in order to accurately and efficiently detect vitamin B6 and its metabolites, it is necessary to explore reagents that can stabilize PLP. The application uses TCA, AA and MA (malic acid) and other organic acids and inorganic acids (such as HCl, acetic acid, carbonic acid) to prepare the corresponding calibration solution and internal standard, and detects the quality control and standard of PLP, and finds that the effect of organic acids is generally better than that of inorganic acids; further, when the solvents are all organic acids, PLP dissolved in malic acid is the most stable, indicating that malic acid has the best effect on preventing PLP from hydrolyzing.

[0009] Malic acid, also known as 2-hydroxysuccinic acid (HOOC-CH(OH)-CH2-COOH), is a naturally occurring organic acid. The metabolism of malic acid (L-malic acid) in the body mainly participates in energy metabolism and material transport through two key pathways: the tricarboxylic acid cycle (TCA cycle) and the malate-aspartate shuttle. Based on the structural characteristics of malic acid, the present invention believes that the reason why it can stabilize PLP is that: first, malic acid is a polycarboxylic acid and an effective metal ion chelator that can form stable chelates with a variety of metal ions (such as iron, copper, etc.). These metal ions often act as catalysts to promote oxidation reactions, thereby leading to the degradation of PLP. By chelating these metal ions, malic acid can reduce the catalytic degradation of PLP by metal ions. Secondly, PLP is relatively stable under acidic conditions, and alkaline environments may accelerate its degradation. Malic acid can regulate the pH of the solution, maintaining an acidic environment (pH 2.4-3.8), which helps protect PLP from degradation. Furthermore, malic acid can act as an antioxidant or antioxidant cofactor, reducing the oxidative degradation of PLP by scavenging free radicals or inhibiting oxidation reactions. In summary, malic acid inhibits PLP degradation through multiple functions.

[0010] It should be understood that the reagents currently commonly used to prepare calibrants cannot achieve the purpose of stabilizing PLP. For example, although BSA has a certain metal chelating ability, it does not have the ability to adjust the pH of the solution and antioxidant capacity. Although PBS can adjust the pH value of the solution, it has no metal chelating ability and antioxidant capacity.

[0011] Furthermore, the vitamin B6 and its metabolites include one or more of PLP, PL, PA, PN, PNP, PM, and PMP.

[0012] It should be understood that although the present invention focuses on the optimization of PLP, PL, and PA detection results, since PN and PNP are relatively stable and do not have degradation issues, and the peak shape and response of PN and PNP are also very good in the presence of malic acid, malic acid can also be used for the detection of PN and PNP.

[0013] In some embodiments, the vitamin B6 and its metabolites include PLP, PL, PA, PN, and PNP.

[0014] In another aspect, the present invention provides a kit for detecting vitamin B6 and its metabolites, wherein the kit comprises a reagent containing malic acid.

[0015] Furthermore, the concentration of the malic acid is 0.1% to 20%, preferably 0.1% to 10%, and more preferably 1%.

[0016] In some aspects, the reagent is any one or more of an internal standard, a standard, a calibrator, a quality control.

[0017] An internal standard is a known amount of a compound (usually similar chemical properties to the analyte but distinguishable in physical and chemical behavior) added in the sample pretreatment stage to correct systematic errors and operational fluctuations in the experiment process, to improve the accuracy and precision of quantitative analysis. Its core use is as follows: correct instrument response fluctuations, compensate for signal fluctuations caused by environmental changes, sample volume differences, etc. in instruments (such as chromatography, mass spectrometry); eliminate sample matrix interference, reduce loss or contamination of the analyte in sample pretreatment (such as extraction, concentration, derivatization); quantitative calculation, quantitative by the ratio of response values (such as peak area ratio) of the analyte, reduce the influence of external factors on the results.

[0018] A standard is a compound with known purity, concentration or characteristics, used to establish a calibration curve for an analytical method or verify the accuracy of a method, which can be divided into working standards, certified reference materials (CRM), primary standards. Its core use is as follows: establish a calibration curve, determine the instrument response value by a series of standard concentrations, establish the relationship between the concentration of the analyte and the signal; method validation, evaluate the linear range, detection limit, precision and accuracy of the method; quality control, monitor the stability of the experimental process (such as batch-to-batch differences).

[0019] In some aspects, the reagent includes an internal standard and a calibrator. In some specific embodiments, the internal standards of PLP, PL, PA, PN, PNP are PLP-d3, PL-d3, PA-d3, PN-d4, PNP-d3, respectively, all of which are deuterated isotopic internal standards, with a concentration of 10-150 nmol / L. In some specific embodiments, the calibrator is set with 7 concentration gradients for preparing a calibration curve.

[0020] In some aspects, the kit further comprises a protein precipitant.

[0021] Further, the protein precipitant comprises one or more of trichloroacetic acid, acetonitrile and methanol.

[0022] TCA precipitation has the characteristics of simple pretreatment operation and complete protein precipitation. Compared with acetonitrile and methanol precipitation, TCA can improve the response of PLP and greatly improve the peak shape of PLP, so TCA is preferred as a protein precipitant. In some specific embodiments, the concentration of TCA is 25%.

[0023] In another aspect, the present application provides a method for detecting vitamin B6 and its metabolites, which refers to using the kit as described above to detect vitamin B6 and its metabolites in a sample.

[0024] In some modes, the steps of the method are as follows: pretreatment of the sample, and then using liquid chromatography-mass spectrometry to detect vitamin B6 and its metabolites in the pretreated sample.

[0025] Further, the pretreatment refers to adding an internal standard solution prepared with malic acid and a protein precipitant to the sample. In some modes, the internal standard is added first, and then the protein precipitant is added; in other modes, the protein precipitant is added first, and then the internal standard is added.

[0026] In some modes, a calibration solution prepared from malic acid is also required to draw a calibration curve.

[0027] The beneficial effects of the present application include:

[0028] 1. The pretreatment step in the method provided by the present application is simple, only needs to add the sample to the internal standard, and then add 25% TCA solution, and after vortex centrifugation, the pretreatment of the sample is completed;

[0029] 2. The present application simultaneously detects five important indicators (PLP, PL, PN, PNP, and PA) related to vitamin B6 in biological samples by liquid chromatography-mass spectrometry, and the sensitivity of liquid chromatography-mass spectrometry is high, so that low-content indicators can also be accurately quantified;

[0030] 3. The method detects multiple indicators, covers important substances in the metabolic pathway of vitamin B6, and can provide more data for doctors in clinical practice, which is beneficial for doctors to accurately judge the development of the patient's condition and improve the diagnosis and treatment efficiency;

[0031] 4. The detection time of the method is fast, only 4 minutes is required to detect one sample, which is more suitable for clinical application;

[0032] 5. Through the results of the preliminary experiment, it is found that as the storage time of the PLP standard water solution increases, the PLP concentration decreases, and the PL and PA concentrations increase. Even if it is stored at -80℃, PLP will still degrade, so the calibration solution needs to be prepared and used, which is complicated and wasteful, and is not suitable for clinical detection. Based on this, the present application provides an acidic environment with less free metal ions for PLP by adding 1% MA, inhibits the degradation of PLP, and makes it stable for more than 6 months;

[0033] 6. By adding 1% malic acid, the degradation problem of PLP is solved, and the accuracy of the detection result is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced. Obviously, the accompanying drawings in the following description only only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0035] Figure 1 : Chromatographic peaks of PLP under different protein precipitants;

[0036] Figure 2 : Mass spectrum detection results of PLP standard aqueous solution after being stored at -80°C for 1 month;

[0037] Figure 3 : Mass spectrum detection results of PLP standard 1% MA solution after being stored at -80°C for 1 month;

[0038] Figure 4 : Chromatogram of PLP, wherein the blue line represents the calibrant and the red line represents the internal standard;

[0039] Figure 5 : Chromatogram of PL, wherein the blue line represents the calibrant and the red line represents the internal standard;

[0040] Figure 6 : Chromatogram of PA, wherein the blue line represents the calibrant and the red line represents the internal standard;

[0041] Figure 7 : Chromatogram of PN, wherein the blue line represents the calibrant and the red line represents the internal standard;

[0042] Figure 8 : Chromatogram of PNP, wherein the blue line represents the calibrant and the red line represents the internal standard;

[0043] Figure 9 : Calibration curve of PLP;

[0044] Figure 10 : Calibration curve of PL;

[0045] Figure 11 : Calibration curve of PA;

[0046] Figure 12 : Calibration curve of PN;

[0047] Figure 13 : Calibration curve of PNP. DETAILED DESCRIPTION

[0048] The application will be further described in conjunction with the accompanying drawings and specific embodiments of the application, which are presented herein for purposes of illustration only and are not intended to be limiting of the scope of the application; based on the embodiments of the application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the application.

[0049] The test methods used in the following examples are conventional methods, all with three replicates, unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials, unless otherwise specified.

[0050] The terms and corresponding English abbreviations involved in the application are as follows:

[0051] TCA: trichloroacetic acid; RBC: red blood cell; PLA: plasma; PLP: pyridoxal phosphate; PL: pyridoxal; PN: pyridoxine; PNP: pyridoxine phosphate; PA: pyridoxal acid; MA: malic acid; AA: ascorbic acid.

[0052] Example 1: A method for detecting vitamin B6 and its metabolites in a blood sample

[0053] 1.1 Preparation of calibrators and internal standards

[0054] PLP, PL, PA, PN, PNP standard solutions were prepared into a mixed solution with deionized water, which was used as a working solution for preparing calibrators, and the standard concentrations of PLP, PL, PA, PN, PNP were 456.90, 149.52, 272.93, 147.93, and 200.80 nmol / L, respectively; then, the above-mentioned standard working solution was gradiently diluted with deionized water to prepare samples (i.e. calibrators) for drawing a calibration curve, and the concentrations are shown in Table 1. The internal standards of PLP, PL, PA, PN, PNP were PLP-d3, PL-d3, PA-d3, PN-d4, and PNP-d3, respectively, and were prepared into an internal standard solution containing 150 nmol / L PLP-d3, 150 nmol / L PL-d3, 50 nmol / L PA-d3, 10 nmol / L PN-d4, and 10 nmol / L PNP-d3, respectively, with deionized water.

[0055] It should be noted that the above-mentioned preparation of calibrators and internal standard solutions with deionized water is a conventional method without improvement. After the application found that PLP cannot be kept stable in aqueous solution for a long time, the solvent of the calibrator and internal standard solution was optimized, which is described in detail in Example 3.

[0056] Table 1: Concentrations of calibrators (S1-S7)

[0057] Calibrator name PLP (nmol / L) PL (nmol / L) PA (nmol / L) PN (nmol / L) PNP (nmol / L) S1 7.13 2.33 4.26 2.31 3.13 S2 14.27 4.67 8.52 4.62 6.27 S3 28.55 9.33 17.09 9.23 12.57 S4 57.11 18.72 34.12 18.52 25.10 S5 114.22 37.38 68.23 36.98 50.20 S6 228.45 74.76 136.46 73.96 100.40 S7 456.90 149.52 272.93 147.93 200.80

[0058] Note: S1 calibrator means the solution containing 7.13 nmol / L PLP, 2.33 nmol / L PL, 4.26 nmol / L PA, 2.31 nmol / L PN and 3.13 nmol / L PNP simultaneously; S2-S7 calibrators are similar.

[0059] 1.2 Pretreatment

[0060] 2 mL whole blood sample was drawn clinically, centrifuged (centrifugation condition: 1,000 g, 5 min), and the supernatant plasma (PLA sample) was drawn. The remaining precipitate was washed with 2 volumes of normal saline, resuspended, and 2 mL or so of red blood cell sample (RBC sample) was prepared.

[0061] 100 μL of stable isotope internal standard solution (containing PLP-d3, PL-d3, PN-d4, PA-d3 and PNP-d3, prepared by the method described above) was added to 80 μL of RBC or PLA sample, followed by 100 μL of 25% TCA. After vortex mixing for 1 min, centrifugation at 12,000 g for 5 min, the supernatant was taken, and the supernatant was detected by liquid chromatography-mass spectrometry.

[0062] 1.3 Sample detection

[0063] Liquid chromatography conditions: the liquid chromatography used a chromatographic column Force C18, 3 μm, 100*30 mm; column temperature: 40 °C; injection volume: 5 μL; flow rate: 0.5 mL / min; mobile phase included: A: water containing 0.1% formic acid, B: acetonitrile; gradient elution conditions were: 0-1.0 min, 4% B; 1.0-1.8 min, 4-95% B; 1.8-2.5 min, 95% B; 2.5-2.7 min, 95-4% B; 2.7-4 min, 4% B; mass spectrometry conditions were shown in Table 2, and ion pair information was shown in Table 3.

[0064] Table 2 Mass spectrometry conditions

[0065] Ion Mode ESI Curtain Gas 35 IonSpray Voltage 5500 Temperature 650 lon Source Gas1 55 lon Source Gas2 55 Collision Gas 7 Scan type MRM

[0066] Table 3 Ion pair information

[0067] Compound Parent ion (m / z) Daughter ion (m / z) De-clustering voltage (volts) Collision energy (volts) PLP 248.0 150.0 60.0 25.0 IS-PLP 251.1 153.1 60.0 25.0 PL 168.0 150.0 40.0 20.0 IS-PL 171.0 153.0 40.0 20.0 PN 170.0 152.0 60.0 20.0 IS-PN 174.1 156.0 60.0 20.0 PNP 250.0 232.1 85.0 19.0 IS-PNP 253.0 235.1 85.0 19.0 PA 184.0 148.0 20.0 30.0 IS-PA 187.1 150.1 20.0 30.0

[0068] 1.4 Detection of calibration curve

[0069] The calibrators S1-S7 in Table 1 were detected by liquid chromatography-mass spectrometry, and the parameters of liquid chromatography were as described above, to obtain the calibration curve.

[0070] Example 2: Selection of precipitant

[0071] In order to further improve the detection efficiency of PLP by liquid chromatography-mass spectrometry, this example screened protein precipitants based on the method described in Example 1, that is, 25% TCA, 100% acetonitrile and 100% methanol were used to pretreat the blood samples. The other steps were the same as those described in Example 1. The experimental results are shown in Tables 4 and Figure 1 .

[0072] Table 4 Selection of protein precipitants

[0073]

[0074]

[0075] The results showed that TCA was the best method for protein precipitation compared with acetonitrile and methanol, because TCA had a stronger precipitation effect, lower residual interferences, good stability and compatibility compared with methanol and acetonitrile, which was specifically reflected in the response and peak shape of PLP.

[0076] Example 3: Optimization of solvent

[0077] In combination with the reagents used in Examples 1 to 2, this example provides a kit for detecting vitamin B6 and its metabolites in blood samples, prepared based on deionized water. The kit comprises a calibrator solution (80 μL / time) containing PLP, PL, PA, PN, and PNP, an internal standard solution (100 μL / time), and a precipitant of 25% TCA (100 μL / time).

[0078] In this example, the kit was stored in a -80 degree Celsius refrigerator. One month later, the kit was subjected to quality control inspection using liquid chromatography-mass spectrometry. After the quality control product stored at -80 degrees Celsius (the quality control product was prepared by 1% BSA, and 20 and 200 μL of 20-fold S7 calibrator solution were added to 10 mL of 1% BSA to prepare QCL and QCH, respectively, and the experimental steps of the detection were as described above) was equilibrated to room temperature, 80 μL of the quality control product was added to the internal standard solution, and then 100 μL of 25% TCA was added. After vortex centrifugation and the supernatant was taken for detection, it was found that the PLP quantitative level was high, while the PL and PA quantitative levels were low. At the same time, the PLP standard (9138 nmol / L) stored in aqueous solution was also detected. It was found that the PLP in the standard was degraded into PL and PA. It was then speculated that the pure aqueous solution could not keep PLP stable ( Figure 2). Based on this, the aqueous solution (solvent) in the kit is replaced with 1% AA (ascorbic acid), 1% TCA (trichloroacetic acid), and 1% MA (malic acid), respectively, that is, 1% AA, 1% TCA, and 1% MA are used to replace the aqueous solution to prepare the calibration solution and the internal standard solution, respectively; and the prepared kit is stored in a -80 degree Celsius refrigerator for one month; after one month, the same stable quality control (as described above) stored at -80 degrees Celsius is detected by using the three kits, respectively, to determine the stability of the kit. The detection results show that AA, TCA, and MA can stabilize PLP to a certain extent, and compared with pure water, 1% AA system, and 1% TCA system, the detection values of PLP, PL, and PA in the 1% MA system kit are basically not deviated from the target value of the quality control, indicating that under the condition of 1% MA, the stability of PLP is the best, in other words, the kit prepared by 1% MA solution significantly inhibits the degradation of PLP, so that the detection results of different forms of vitamin B6 concentration are more accurate (Table 5); in addition, this example also proves that the PLP standard prepared by 1% MA solution is not degraded into PL and PA after being stored at -80 degrees Celsius for one month, indicating that the stability of PLP is high Figure 3 ). In summary, MA (the preferred concentration in this example is 1%) can prevent PLP from being converted into PL and PA, and the mechanism of action of MA is as follows:

[0079] 1. Buffering effect

[0080] Malic acid (MA) is an organic acid with certain buffering capacity, which can stabilize the pH value of the solution. PLP is easily degraded (such as hydrolysis or photolysis) under alkaline or strong acidic conditions, while malic acid can keep PLP in a weak acidic environment (pH 2-5) for a long time, thereby reducing the chemical decomposition of PLP.

[0081] 2. Chelating metal ions

[0082] Metal ions (such as Fe 2+ , Cu 2+ ) can catalyze the degradation reaction of PLP, at the same time, these ions can also destroy the structure of PLP through oxidation reaction or complexation. While malic acid contains carboxyl and hydroxyl groups, which can chelate metal ions, reduce their catalytic activity, and thus play a protective role for PLP.

[0083] 3. Antioxidant effect

[0084] Malic acid has certain reducing property, which can scavenge free radicals and inhibit oxidation reaction, preventing PLP from being converted into other substances due to oxidation, such as the aldehyde group of PLP being oxidized into the inactive carboxylic acid form.

[0085] In addition, the present embodiment also attempts to use inorganic acids (including hydrochloric acid, acetic acid and carbonic acid) to prepare the calibration solution and the internal standard solution, and it is found that the effect of inorganic acid is generally worse than that of organic acid, and even the degradation of PLP is accelerated, which may be because the buffering capacity of inorganic acid is not as good as that of organic acid, and hydrochloric acid and acetic acid are easy to volatilize, carbonic acid is easy to degrade and has weak ionization capacity.

[0086] Therefore, in order to inhibit the conversion of PLP into other substances and prolong the shelf life of the kit for detecting vitamin B6 and its metabolites, it is preferred to use organic acid to prepare the calibration solution and the internal standard solution containing PLP, and more preferably, MA; the preparation method of the two solutions is described in Example 1.

[0087] Table 5 - Quality control test results of different kits after 1 month of storage at 80°C

[0088]

[0089] Example 4 - Optimization of the concentration of malic acid

[0090] Through the screening of Example 3, the substance that can most inhibit the decomposition of PLP is obtained, i.e. malic acid. In order to further improve the inhibitory effect of malic acid on the decomposition of PLP, the present embodiment explores the influence of the concentration of malic acid on the degradation rate of PLP.

[0091] In the present embodiment, the internal standard solution and the calibration solution are prepared by using 0.1%, 0.5%, 1%, 5% and 10% MA solution respectively, and the kit is stored in a refrigerator at -80°C. After one month, the kit is subjected to quality control test by liquid chromatography mass spectrometry, and the test steps are the same as described in Example 3. The specific results are shown in Table 6. The results show that, compared with the 0.1%, 0.5%, 5% and 10% MA systems, the detection values of PLP, PL and PA in the 1% MA system kit have the smallest deviation from the quality control target value, indicating that the stability of PLP is best under the condition of 1% MA. This may be because when the concentration of MA is less than 1%, the low concentration of MA cannot completely chelate the metal ions in the solution, and thus cannot inhibit the degradation of PLP; and as the concentration of MA increases (>1%), the pH value of the solution decreases, and thus the buffering effect is not good and the degradation of PLP cannot be inhibited. Therefore, the concentration of malic acid is preferably 1% to 5%, and more preferably 1%.

[0092] Based on the results of Examples 2-4, the present application provides a new 1% MA system kit for detecting vitamin B6 and its metabolites, which comprises the following components: a calibration solution prepared with 1% MA solution and containing PLP, PL, PA, PN and PNP (the concentrations of the calibration are shown in Table 1), an isotopic internal standard solution prepared with 1% MA solution and containing five vitamin B6 and its metabolites (the concentrations of the internal standard are shown in Example 1), and 25% TCA, and the method for using the kit is described in Example 1.

[0093] Table 6 - Quality control test results of the kit with different MA concentrations after being stored at 80°C for 1 month

[0094]

[0095] Example 5 - Performance test of the 1% MA system kit

[0096] Examples 2-4 respectively optimize the precipitant and solvent in the kit for detecting vitamin B6 and its metabolites, and then a new 1% MA system-TCA precipitant kit is obtained. The kit is used to detect PLP, PL, PA, PN and PNP at concentrations of 386, 113, 165, 86 and 124 nmol / L respectively (the specific experimental steps are described in Example 1), and the corresponding chromatograms are shown in Figure 4-8 As can be seen from the figure, the chromatographic peaks of each substance are symmetrical, the baseline is low, and the sensitivity is high, indicating that the liquid chromatography-mass spectrometry method is accurate and reliable. In addition, the overall performance of the 1% MA kit is also verified in this example.

[0097] 5.1 Linearity

[0098] 100 μL of 25% TCA is added to the calibration groove of the kit (which already contains 80 μL of calibration and 100 μL of internal standard), vortexed for 1 min, then centrifuged at 2,000 g for 15 min, and the supernatant was injected for detection.

[0099] As can be seen from the figure, the chromatographic peaks of each substance are symmetrical, the baseline is low, and the sensitivity is high, indicating that the liquid chromatography-mass spectrometry method is accurate and reliable. In addition, the overall performance of the 1% MA kit is also verified in this example. Figure 9-13 It can be seen that the linear correlation coefficients R 2 of the five detection substances are all greater than 0.999, indicating that there is a very strong linear relationship between the concentrations of the five detection substances and the response values, and the linear regression model established has a very high fitting degree for the experimental data, which provides a reliable mathematical basis for the quantitative analysis of the substances. At the same time, this also reflects the consistency and repeatability of the experimental data, as well as the strict control of the experimental operation and measurement process.

[0100] 5.2 Lower limit of detection

[0101] The LLOQ (Lower Limit of Quantification) samples of each target substance were determined, and 6 samples were determined in parallel. 80 μL of the LLOQ sample was added to 100 μL of the internal standard of 1% MA, and then 100 μL of 25% TCA was added. After vortex mixing for 1 min, centrifugation was performed at 2000 g for 15 min, and the supernatant was taken for sample detection. The specific results are shown in Tables 7-11. The signal-to-noise ratio of the measured substance and the internal standard chromatographic peak in the LLOQ concentration level standard sample chromatogram of each substance was not less than 5, the deviation of the measured value from the theoretical value was within ± 20.0%, and the coefficient of variation was not more than 15%. Therefore, the lower limits of quantification of PLP, PL, PA, PN and PNP were 7.13, 2.33, 4.26, 2.31 and 3.13 nmol / L, respectively.

[0102] Table 7 Lower limit of quantification of PLP

[0103]

[0104] Table 8 Lower limit of quantification of PL

[0105]

[0106] Table 9 Lower limit of quantification of PA

[0107]

[0108] Table 10 Lower limit of quantification of PN

[0109]

[0110] Table 11 Lower limit of quantification of PNP

[0111]

[0112] 5.3 Precision

[0113] Low and high concentration quality control samples were used as samples for detection (detection steps are as described above), in order to investigate the precision of the analysis batch and the analysis batch. Each sample was processed in parallel for 6 times, and the specific results are shown in Tables 12-16. The repeatability (CV) of the low and high concentration level quality control samples was less than 15.0%. Therefore, the inter-batch precision and intra-batch precision of the kit meet the requirements.

[0114] Table 12 Precision of detection of PLP

[0115]

[0116]

[0117] Table 13 Precision of detection of PL

[0118]

[0119]

[0120] Precision of PA detection

[0121]

[0122] Precision of PN detection

[0123]

[0124]

[0125] Precision of PNP detection

[0126]

[0127]

[0128] 5.4 Matrix effect

[0129] Select 3 different sources of human sample matrix (plasma and red blood cells), compare the peak area of 3 different sources of human sample matrix after adding different concentration levels and pure solvent, calculate the matrix factor of each analyte and internal standard by calculating the analyte area B (measured after adding analyte after treating 3 different sources of human sample matrix), internal standard peak area B IS in the presence of matrix, analyte background peak area A (measured after treating 3 different sources of human sample matrix), internal standard peak area A IS in the presence of matrix, analyte area C (measured after adding analyte after treating water solution), internal standard peak area C IS without matrix. Further, by dividing the matrix factor of the analyte by the matrix factor of the internal standard, the matrix factor normalized by the internal standard is calculated, and the matrix factor should be between 0.85 and 1.15. The specific results are shown in Table 17. The results show that the matrix factors of the plasma sample and the red blood cell sample are both between 0.85 and 1.15, meaning that the matrix has little effect on the response of the analyte, and the analysis result is reliable; and the analysis method is suitable for the detection of plasma and red blood cell samples, without the need for complex matrix correction.

[0130] Matrix effect

[0131]

[0132]

[0133] 5.5 Stability

[0134] In view of practical application, the long-term storage stability of the kit of the 1% MA system is investigated in this embodiment, that is, the kit is stored at-20 degrees Celsius, and after 0, 3, 6 months, the kit is taken out and used to detect the stable quality control samples stored at-80 degrees Celsius, and the specific results are shown in Tables 18-22. The results show that the deviation of the quality control detection values of the kit for 6 months from the theoretical values is within ±20.0%, and the CV is within 15%, indicating that the kit of the 1% MA system can be stored at-20 degrees Celsius for at least 6 months.

[0135] Table 18 Stability of detection of PLP

[0136]

[0137] Table 19 Stability of detection of PL

[0138]

[0139]

[0140] Table 20 Stability of detection of PA

[0141]

[0142] Table 21 Stability of detection of PN

[0143]

[0144]

[0145] Table 22 Stability of detection of PNP

[0146]

[0147] In summary, the kit for detecting vitamin B6 and its metabolites provided by the present application has better overall performance, and the accuracy and repeatability of the experimental results are higher.

[0148] Example 6: A new kit for detecting vitamin B6 and its metabolites and a method of using the same

[0149] According to all the experimental results of Examples 1-5, this embodiment provides a new kit for detecting vitamin B6 and its metabolites, which comprises the following components:

[0150] A. calibrators: the calibrators are divided into 7 concentrations (i.e. S1-S7, the specific concentrations are shown in Table 1), and are dissolved in 1% MA solution, that is, the solvent of the calibrator solution is 1% MA;

[0151] B. Internal standard solution: The internal standard solution contains PLP, PL, PA, PN, and PNP simultaneously; the internal standards of PLP, PL, PA, PN, and PNP are PLP-d3, PL-d3, PA-d3, PN-d4, and PNP-d3, respectively, and the concentrations of the five substances are 150, 150, 50, 10, and 10 nmol / L, respectively, and the solvent of the internal standard solution is 1% MA;

[0152] C. Protein precipitant: 25% TCA.

[0153] The steps for using the above kit are as follows: pre-treat the blood sample with protein precipitant 25% TCA, perform liquid chromatography-mass spectrometry on the treated sample, run the calibration standard of each concentration to obtain the calibration curve ( Figure 9-13 ), and the test results of the blood samples were substituted into the standard curve to obtain the corresponding concentrations of vitamin B6 and its metabolites. The specific experimental steps were the same as those described in Example 1.

[0154] Furthermore, experiments have shown no difference in the effectiveness of the aforementioned kits when testing different samples (RBC or PLA), meaning that this method can be used to test both red blood cell and plasma samples. It is important to understand that due to differences in the distribution of various substances in red blood cells and plasma, separate testing of substance concentrations in red blood cells and plasma can avoid cross-interference and more accurately reflect physiological or pathological states, particularly in the assessment of nutrition, metabolic diseases, and hematological disorders.

[0155] References

[0156] [1] Roelofsen-de Beer R, Van Zelst BD, Wardle R, et al. Simultaneous measurement of whole blood vitamin B1 and vitamin B6 using LC-ESI-MS / MS[J]. Journal of Chromatography B, 2017, 1063: 67-73.

[0157] The foregoing presents and describes the basic principles and main features of the present application and the advantages thereof, it being apparent that the present application is not limited to the details of the above-described exemplary embodiments, but that it can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application, and that it is therefore intended to cover all the changes and modifications which fall within the meaning and scope of the equivalent elements of the claims, the claims being intended to cover all the equivalents of the product and method and the scope of protection of the present application being defined by the claims, it being intended, therefore, that all the changes and modifications which fall within the meaning and scope of the equivalent elements of the claims be embraced therein, any reference signs in the claims not being considered limiting of the claims in question.

Claims

1. Use of organic acids for preparing stable pyridoxal phosphate and / or detecting vitamin B6 and its metabolite preparations.

2. The use according to claim 1, characterized in that The vitamin B6 and its metabolites include one or more of PLP, PL, PA, PN, PNP, PM, and PMP.

3. A kit for detecting vitamin B6 and its metabolites, characterized in that: The kit comprises a reagent comprising malic acid.

4. The kit according to claim 3, wherein The concentration of the malic acid is 0.1% to 20%.

5. The kit according to claim 3, wherein The reagents are any one or more of internal standards, quality control products, calibrators, and standard products.

6. The kit according to claim 3, wherein The kit also includes a protein precipitation agent.

7. The kit according to claim 6, wherein The protein precipitant comprises one or more of trichloroacetic acid, acetonitrile and methanol.

8. A method for detecting vitamin B6 and its metabolites, characterized in that: The method refers to using the kit according to any one of claims 3 to 7 to detect vitamin B6 and its metabolites in a sample.

9. The method according to claim 8, wherein The method comprises the following steps: pre-treating the sample, and then using liquid chromatography-mass spectrometry to detect vitamin B6 and its metabolites in the treated sample.

10. The method according to claim 9, wherein The pretreatment refers to adding an internal standard solution prepared with malic acid and a protein precipitant to the sample.

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