A method for quantitatively detecting vitamin K3

Abstract

The application is a method for quantitatively detecting vitamin K3, characterized in that: a "HCHO NaHSO3-Na2SO3" pH clock reaction system is used as a detection solution, and different responses of the system to different concentrations of vitamin K3, namely different induction times, are used to realize quantitative analysis of vitamin K3. The quantitative analysis method for vitamin K3 disclosed by the application has the characteristics of high accuracy, easy operation, and convenience and rapidness.

Description

Technical Field

[0001] This invention relates to an analytical detection method, specifically, the establishment of "HCHO" A pH clock system using "-NaHSO3-Na2SO3" as substrates is used to achieve quantitative analysis of vitamin K3 based on the different responses of this system to different concentrations of vitamin K3, i.e., the different induction times. This method belongs to the field of analytical chemistry. Background Technology

[0002] Vitamin K3, molecular formula C 11 H8O2 is an analytical reagent used in chemical analysis. Vitamin K3 is primarily a procoagulant, used to treat hemorrhagic diseases caused by vitamin K deficiency, such as neonatal hemorrhage, vitamin K deficiency due to intestinal malabsorption, and hypoprothrombinemia. Vitamin K3 is a synthetically produced form of vitamin K. As an essential substance for physiological blood clotting, vitamin K participates in the synthesis of clotting factors and maintains the physiological process of blood coagulation in the human body.

[0003] Currently, methods for detecting vitamin K3 include liquid chromatography, spectrophotometry, and electrochemical methods. However, most of these methods require large equipment and are expensive, making them unsuitable for on-site testing. Therefore, it is essential to find a detection and analysis method that is effective, easy to operate, and rapid. The structure of vitamin K3 is shown in structural formula (Ⅰ).

[0004] The structural formula of vitamin K3 (I) Summary of the Invention

[0005] This invention aims to provide a new quantitative detection method for vitamin K3, namely, using "HCHO" The "-NaHSO3-Na2SO3" pH clock system is a method for the quantitative detection of vitamin K3 in a detection solution. This method is a standard curve (working curve) method developed based on the sensitive response of this pH clock system to vitamin K3. Specifically, it applies "HCHO" The "-NaHSO3 - Na2SO3" pH clock reaction system is used as the detection solution to record the pH change over time. When the pH clock reaction starts, equal volumes of a series of vitamin K3 sample solutions of different concentrations are added to the pH clock system. Based on the different induction times generated by the system when the concentration of the sample solution in the pH clock system is different, the quantitative detection of the vitamin K3 sample can be achieved.

[0006] A working curve was established based on the relationship between the concentration of vitamin K3 in the pH clock system and the induction time; the horizontal axis represents the concentration of vitamin K3 in the pH clock system, and the vertical axis represents the induction time t. When the concentration of vitamin K3 in the system is 7.25 × 10⁻⁶, the curve is used to establish the working curve. -5 mol / L to 5.08×10 -4 When the concentration is between mol / L, the induction time t has a linear relationship with the concentration of vitamin K3, which allows for the quantitative detection of vitamin K3 in the sample.

[0007] The difference between this quantitative detection method and existing technologies lies in the application of "HCHO". The pH clock system of "NaHSO3-Na2SO3" is used as the detection solution, and the different responses of this system to different concentrations of vitamin K3, i.e., the different induction times, enable the quantitative analysis of vitamin K3.

[0008] The detectable concentration range of vitamin K3 in the detection solution (pH clock system) is 7.25 × 10⁻⁶. -5 -5.08×10 -4 mol / L.

[0009] When vitamin K3 is detected in the detection solution (pH clock system), the temperature of the pH clock system is controlled at any specific temperature within the range of 10-18℃.

[0010] Using the aforementioned pH clock system, the detectable concentration range of vitamin K3 is the optimal range determined experimentally. Within this concentration range, the induction time responds well to changes in vitamin K3 concentration, exhibiting a large linear correlation coefficient. Furthermore, the concentration ranges of each component in the detection solution (pH clock system) are shown in Table 1, and the optimal concentrations of the detection solution (pH clock system) obtained through multiple experiments are shown in Table 2.

[0011] Table 1: Concentration of each component in the pH clock system

[0012] HCHO (mol / L) <![CDATA[NaHSO 3 (mol / L)]]> <![CDATA[Na2SO3 (mol / L)]]> 0.043-0.0635 0.035-0.0625 0.0035-0.00625

[0013] Table 2: Optimal concentrations of each component in the pH clock system

[0014] HCHO (mol / L) <![CDATA[NaHSO 3 (mol / L)]]> <![CDATA[Na2SO3 (mol / L)]]> 0.0501 0.0499 0.00499

[0015] The specific experimental steps are as follows:

[0016] 1. Prepare a 40 mL test solution (pH clock system) according to the concentration range specified in Table 1, maintaining its temperature at a specific value between 10-18℃. Insert the prepared working electrode (pH composite electrode, Leici, E-331) into the solution. Connect the other end of the working electrode to a computer via a potential / temperature / pH integrated tester (Jiaxing Disheng Electronic Technology Co., Ltd., ZHFX-595). Open the chemical signal acquisition and analysis program on the computer, set the acquisition time and sampling speed, and quickly click the start button to monitor the pH of the solution. The computer records the collected pH change curve over time, i.e., the pH clock spectrum. When a substance needs to be detected, add the analyte immediately after the pH clock system reaction begins, and record the pH change over time in the same manner.

[0017] The basic parameters of a pH clock spectrum include:

[0018] Induction time: The time required from the start of the reaction in the pH clock system to the pH jump.

[0019] pH jump range: from the pH at which the pH jump begins to the pH at which the pH jump ends.

[0020] Establish a working curve to determine the relationship between vitamin K3 concentration in the detection solution and pH induction time.

[0021] Vitamin K3 solutions with concentrations ranging from 0.0725 mol / L to 0.508 mol / L were prepared using ethanol as the solvent. Simultaneously with the start of the pH clock system reaction, 40 μL of each of these different concentrations of sample solution was added to a 40 mL pH clock system using a pipette, resulting in a vitamin K3 concentration of 7.25 × 10⁻⁶. -5 mol / L to 5.08×10 -4 The pH clock system response changes within the range of mol / L; the induction time is denoted as t. The induction time t varies with different vitamin K3 concentrations in the system. A graph is plotted with vitamin K3 concentration on the x-axis and t on the y-axis. When the vitamin K3 concentration is between 7.25 × 10⁻⁶ mol / L... -5 mol / L to 5.08×10 -4 When the concentration of vitamin K3 is between mol / L, the induction time t of the pH clock system has a linear relationship with the concentration of vitamin K3, and the working curve is obtained.

[0022] Quantitative detection of vitamin K3

[0023] When a test sample of unknown concentration is added to the pH clock system at the start of the pH clock system reaction, the induction time (t) of the corresponding pH clock system can be measured. Based on the correspondence between t and the unknown vitamin K3 concentration on the working curve, the concentration of vitamin K3 in the detection system can be obtained, and then the concentration of vitamin K3 in the test sample can be calculated. Attached Figure Description

[0024] Figure 1 This is a graph showing the change in pH value of the detection solution (pH clock system) over time when no sample to be tested was added, as described in Example 1.

[0025] Figure 2 In Example 1, 7.25 × 10 -5 After adding mol / L vitamin K3, the pH value of the solution (pH clock system) was measured as a function of time.

[0026] Figure 3 In Example 1, 1.45 × 10⁻⁶ was added. -4 After adding mol / L vitamin K3, the pH value of the solution (pH clock system) was measured as a function of time.

[0027] Figure 4 This is the working curve of pH induction time t versus vitamin K3 concentration in Example 1.

[0028] Figure 5 This is a graph showing the change in pH value of the detection solution (pH clock system) over time when no sample to be tested was added, as shown in Example 2.

[0029] Figure 6 In Example 2, 2.9 × 10⁻⁶ was added. -4 After adding mol / L vitamin K3, the pH value of the solution (pH clock system) was measured as a function of time.

[0030] Figure 7 In Example 2, 4.35 × 10⁻⁶ was added. -4 After adding mol / L vitamin K3, the pH value of the solution (pH clock system) was measured as a function of time.

[0031] Figure 8 This is the working curve of pH induction time t versus vitamin K3 concentration in Example 2.

[0032] Figure 9 This is a graph showing the change in pH value of the test solution (pH clock system) over time when no sample to be tested was added, as shown in Example 3.

[0033] Figure 10 In Example 3, 4.35 × 10⁻⁶ was added.-4 After adding mol / L vitamin K3, the pH value of the solution (pH clock system) was measured as a function of time.

[0034] Figure 11 In Example 3, 5.08 × 10⁻⁶ was added. -4 After adding mol / L vitamin K3, the pH value of the solution (pH clock system) was measured as a function of time.

[0035] Figure 12 This is the working curve of pH induction time t versus vitamin K3 concentration in Example 3. Implementation

[0036] Example

[0037] Application with "HCHO" A pH clock system using "-NaHSO3-Na2SO3" as the substrate was used as the detection solution for quantitative analysis of vitamin K3. Equal volumes of vitamin K3 sample solutions of different concentrations were added to the pH clock system to establish a working curve (e.g., a linear relationship) relating vitamin K3 concentration to induction time, thus achieving the purpose of detecting vitamin K3 in the pH clock system and calculating the concentration of vitamin K3 in the test sample.

[0038] (1) Preparation of detection solution

[0039] First, prepare mixed solutions of 0.12 mol / L HCHO, 0.15 mol / L NaHSO3, and 0.015 mol / L Na2SO3 using distilled water. Then, add 10.0 mL of distilled water, 13.3 mL of the NaHSO3-Na2SO3 mixed solution, and 16.7 mL of 0.12 mol / L HCHO solution to a 50 mL beaker sequentially to ensure the HCHO concentration is within the specified range. The concentrations of each component in the "NaHSO3-Na2SO3" pH clock system are 0.0501 mol / L HCHO, 0.0499 mol / L NaHSO3, and 0.00499 mol / L Na2SO3, with a total volume of 40 mL and a temperature controlled at 12℃.

[0040] Meanwhile, a series of vitamin K3 sample solutions of different concentrations were prepared using ethanol as a solvent.

[0041] (2) Obtain pH clock spectrum

[0042] The pH value of the prepared test solution over time was plotted by a computer equipped with a chemical signal acquisition and analysis program (without the test sample added). For example... Figure 1As shown in the figure. The pH induction time was 230 s as a blank control. Two additional test solutions with the same component concentrations as the above test solutions were prepared. For one of these solutions, at the start of the reaction, 40 μL of 0.0725 mol / L vitamin K3 sample solution was added to a 40 mL pH clock system, so that the concentration of vitamin K3 in the test solution was 7.25 × 10⁻⁶. -5 The addition of vitamin K3 at a concentration of mol / L shortened the induction time to 206 s. Figure 2 As shown; for the other group, at the start of the reaction, 40 μL of 0.145 mol / L vitamin K3 sample solution was added to a 40 mL pH clock system, so that the concentration of vitamin K3 in the detection solution was 1.45 × 10⁻⁶. -4 The addition of vitamin K3 at a concentration of mol / L changed the induction time to 187 s. Figure 3 As shown. Figure 2 , Figure 3 This confirmed that different concentrations of vitamin K3 in the detection solution led to different induction times in the pH clock system. When the concentration of vitamin K3 in the detection system was 7.25 × 10⁻⁶, the induction time was significantly different. -5 mol / L to 5.08×10 -4 When the concentration is between mol / L, different concentrations can lead to different induction times in the pH clock system, which can be observed.

[0043] (3) Quantitative detection

[0044] A working curve was established based on the relationship between the concentration of vitamin K3 in the detection system and the induction time, such as... Figure 4 As shown, the horizontal axis represents the concentration of vitamin K3 in the pH clock system, and the vertical axis represents the induction time t. When the concentration of vitamin K3 in the detection system is 7.25 × 10⁻⁶, the value is determined by the pH clock system. -5 mol / L to 5.08×10 -4 When the concentration of vitamin K3 is between mol / L, the induction time has a linear relationship with the concentration of vitamin K3, and the linear equation is t = -244032c(vitamin K3) + 222.57, R 2 =0.997. Based on this, quantitative detection of vitamin K3 in the sample can be achieved.

[0045] Example

[0046] (1) Preparation of detection solution

[0047] First, prepare mixed solutions of 0.12 mol / L HCHO, 0.15 mol / L NaHSO3, and 0.015 mol / L Na2SO3 using distilled water. Then, add 10 mL of distilled water, 14 mL of the NaHSO3-Na2SO3 mixed solution, and 16 mL of 0.12 mol / L HCHO solution to a 50 mL beaker sequentially to ensure the HCHO concentration is within the specified range. The concentrations of each component in the "NaHSO3-Na2SO3" pH clock system are 0.048 mol / L HCHO, 0.0525 mol / L NaHSO3, and 0.00525 mol / L Na2SO3, with a total volume of 40 mL and a temperature controlled at 12℃.

[0048] Meanwhile, a series of vitamin K3 sample solutions of different concentrations were prepared using ethanol as a solvent.

[0049] (2) Obtain pH clock spectrum

[0050] The pH value of the prepared test solution over time was recorded by a computer equipped with a chemical signal acquisition and analysis program (without the test sample added). Figure 5 As shown. The pH induction time was 231 s as a blank control. Two additional test solutions with the same component concentrations as the above test solutions were prepared. For one of these solutions, at the start of the reaction, 40 μL of 0.29 mol / L vitamin K3 sample solution was added to a 40 mL pH clock system, making the vitamin K3 concentration in the test solution 2.9 × 10⁻⁶. -4 The addition of vitamin K3 at a concentration of mol / L shortened the induction time to 151 s. Figure 6 As shown; for the other group, at the start of the reaction, 40 μL of 0.435 mol / L vitamin K3 sample solution was added to a 40 mL pH clock system, so that the concentration of vitamin K3 in the detection solution was 4.35 × 10⁻⁶. -4 The addition of vitamin K3 at a concentration of mol / L changed the induction time to 113 s. Figure 7 As shown. Figure 6 , Figure 7 This confirmed that different concentrations of vitamin K3 in the detection solution led to different induction times in the pH clock system. When the concentration of vitamin K3 in the detection system was 7.25 × 10⁻⁶, the induction time was significantly different. -5 mol / L to 5.08×10 -4 Different concentrations (mol / L) can lead to different induction times in the pH clock system, and these differences can be observed.

[0051] (3) Quantitative detection

[0052] A working curve was established based on the relationship between the concentration of vitamin K3 in the detection system and the induction time, such as... Figure 8 As shown, the horizontal axis represents the concentration of vitamin K3 in the pH clock system, and the vertical axis represents the induction time t. When the concentration of vitamin K3 in the detection system is 7.25 × 10⁻⁶, the value is determined by the pH clock system. -5 mol / L to 5.08×10 -4 When the concentration of vitamin K3 is between mol / L, the induction time has a linear relationship with the concentration of vitamin K3, and the linear equation is t = -240894c(vitamin K3) + 221.05, R. 2 =0.9951. Based on this, quantitative detection of vitamin K3 in the sample can be achieved.

[0053] Example

[0054] (1) Preparation of detection solution

[0055] First, prepare mixed solutions of 0.12 mol / L HCHO, 0.15 mol / L NaHSO3, and 0.015 mol / L Na2SO3 using distilled water. Then, add 10.2 mL of distilled water, 13.2 mL of the NaHSO3-Na2SO3 mixed solution, and 16.6 mL of 0.12 mol / L HCHO solution to a 50 mL beaker sequentially to ensure the HCHO concentration is within the specified range. The concentrations of each component in the "NaHSO3-Na2SO3" pH clock system are 0.0498 mol / L HCHO, 0.0495 mol / L NaHSO3, and 0.00495 mol / L Na2SO3, with a total volume of 40 mL and a temperature controlled at 12℃.

[0056] Meanwhile, a series of vitamin K3 sample solutions of different concentrations were prepared using ethanol as a solvent.

[0057] (2) Obtain pH clock spectrum

[0058] The pH value of the prepared test solution over time was plotted by a computer equipped with a chemical signal acquisition and analysis program (without the test sample added). For example... Figure 9 As shown in the figure. The pH induction time was 230.6 s as a blank control. Two additional test solutions with the same component concentrations as the above test solutions were prepared. For one of these solutions, at the start of the reaction, 40 μL of 0.435 mol / L vitamin K3 sample solution was added to a 40 mL pH clock system, so that the concentration of vitamin K3 in the test solution was 4.35 × 10⁻⁶. -4 The addition of vitamin K3 at a concentration of mol / L shortened the induction time to 113 s. Figure 10As shown; for the other group, at the start of the reaction, 40 μL of 0.508 mol / L vitamin K3 sample solution was added to a 40 mL pH clock system, so that the concentration of vitamin K3 in the detection solution was 5.08 × 10⁻⁶. -4 The addition of vitamin K3 at a concentration of mol / L changed the induction time to 102 s. Figure 11 As shown. Figure 10 , Figure 11 This confirmed that different concentrations of vitamin K3 in the detection solution led to different induction times in the pH clock system. When the concentration of vitamin K3 in the detection system was 7.25 × 10⁻⁶, the induction time was significantly different. -5 mol / L to 5.08×10 - 4 When the concentration is between mol / L, different concentrations can lead to different induction times in the pH clock system, which can be observed.

[0059] (3) Quantitative detection

[0060] A working curve was established based on the relationship between the concentration of vitamin K3 in the detection system and the induction time, such as... Figure 12 As shown, the horizontal axis represents the concentration of vitamin K3 in the pH clock system, and the vertical axis represents the induction time t. When the concentration of vitamin K3 in the detection system is 7.25 × 10⁻⁶, the value is determined by the pH clock system. -5 mol / L to 5.08×10 -4 When the concentration of vitamin K3 is between mol / L, the induction time has a linear relationship with the concentration of vitamin K3, and the linear equation is t = -242971c(vitamin K3) + 222.7, R 2 =0.9963. Based on this, quantitative detection of vitamin K3 in the sample can be achieved.

Claims

1. A quantitative detection method of vitamin K3, characterized in that: a solution of a sample to be detected is prepared with ethanol as a solvent; a "HCHO-NaHSO3-Na2SO3" pH clock reaction system is used as a detection solution, and a graph of pH change with time is recorded; the temperature of the pH clock reaction system is controlled at any specific temperature within the range of 10-18℃, when the pH clock reaction system starts to react, a series of different concentrations of the sample solution to be detected are added into the pH clock reaction system in equal volumes, and the quantitative detection of the sample to be detected is realized according to the different induced time generated by the system when the concentration of the sample solution to be detected in the pH clock reaction system is different; the induced time is the time required from the start of the reaction of the pH clock reaction system to the pH jump; the molar concentration of each component in the detection solution is in the range of HCHO 0.043-0.0635 mol / L, NaHSO3 0.035-0.0625 mol / L, and Na2SO3 0.0035-0.00625 mol / L; the sample to be detected is a vitamin K3 solution; the molar concentration of each component in the detection solution is HCHO 0.0501 mol / L, NaHSO3 0.0499 mol / L, and Na2SO3 0.00499 mol / L. The temperature of the pH clock reaction system for detecting the vitamin K3 solution is controlled at 12℃. ​ ​ ​ A working curve is established according to the relationship between the concentration of the sample solution to be detected in the pH clock reaction system and the induction time; wherein the abscissa is the concentration of vitamin K3 in the sample solution to be detected in the pH clock reaction system, and the ordinate is the induction time t; when the concentration of vitamin K3 in the system is between 7.25×10 -5 mol / L to 5.08×10 -4 mol / L, there is a linear relationship between the induction time t and the concentration of vitamin K3, and accordingly the quantitative detection of vitamin K3 in the sample is realized.

2. The quantitative detection method according to claim 1, characterized in that: ​ 3. The method of claim 1, wherein: ​

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