CN116660338A - Method for quantitatively detecting vitamin K3 - Google Patents

Abstract

The invention relates to a method for quantitatively detecting vitamin K3, which is characterized by comprising the following steps of: application of HCHO-NaHSO ₃ ‑Na ₂ SO ₃ The pH clock reaction system is used as a detection solution, and quantitative analysis of the vitamin K3 is realized according to different responses of the system to different concentrations of the vitamin K3, namely different induction times. The quantitative analysis method for vitamin K3 has the characteristics of high accuracy, easiness in operation, convenience, quickness and the like.

Description

A method for quantitative detection of vitamin K3

technical field

The present invention relates to an analysis and detection method, specifically to establish a pH clock system with "HCHO-NaHSO ₃ -Na ₂ SO ₃ " as a substrate, and according to the different responses of the system to different concentrations of vitamin K3, that is, the difference in induction time The invention relates to a quantitative analysis method for vitamin K3, which belongs to the field of analytical chemistry.

Background technique

Vitamin K3, molecular formula C ₁₁ H ₈ O ₂ , is an analytical reagent in chemical analysis. Vitamin K3 is mainly a procoagulant drug, which can be used for bleeding disorders caused by vitamin K deficiency, such as neonatal hemorrhage, vitamin K deficiency caused by intestinal malabsorption, and hypothrombinogen syndrome. Vitamin K3 is artificially synthesized vitamin K. As a substance necessary for human physiological coagulation, vitamin K participates in the synthesis of coagulation factors and can maintain the physiological process of blood coagulation in the human body.

Current detection methods for vitamin K3 include liquid chromatography, spectrophotometry, and electrochemical methods. However, most of these detection methods require large equipment and are expensive to test, so they are not suitable for on-site determination. Therefore, it is very necessary to find a detection and analysis method with good detection effect and simple and fast operation. The structure of vitamin K3 is shown in structural formula (I).

The structural formula of vitamin K3 (Ⅰ)

Contents of the invention

The present invention aims to provide a new quantitative detection method for vitamin K3, that is, a method for quantitative detection of vitamin K3 using the "HCHO-NaHSO ₃ -Na ₂ SO ₃ " pH clock system as the detection solution. This method is based on the pH A standard curve (working curve) method developed for the sensitive response of the clock system to vitamin K3. Specifically, the "HCHO-NaHSO ₃ -Na ₂ SO ₃ "pH clock reaction system was used as the detection solution, and the spectrum of pH changes with time was recorded; when the pH clock reaction started, a series of different concentrations of vitamin K3 samples to be detected were respectively The equal volume of the solution is added to the pH clock system, and the quantitative detection of the vitamin K3 sample to be detected is realized according to the difference in the induction time generated by the system when the concentration of the solution to be tested is different in the pH clock system.

Establish a working curve according to the relationship between the concentration of vitamin K3 in the pH clock system and the induction time; where the abscissa is the concentration of vitamin K3 in the pH clock system, and the ordinate is the induction time t, when the vitamin K3 concentration in the system is 7.25 × 10 Between ^-5 mol/L and 5.08×10 ^-4 mol/L, the induction time t has a linear relationship with the concentration of vitamin K3, and the quantitative detection of vitamin K3 in the sample can be realized accordingly.

The difference between this quantitative detection method and the prior art is that the present invention uses the "HCHO-NaHSO ₃ -Na ₂ SO ₃ "pH clock system as the detection solution, and the response of the system to different concentrations of vitamin K3 is different, that is, the induction time is different , to achieve quantitative analysis of vitamin K3.

The detected concentration range of vitamin K3 in the detection solution (pH clock system) is 7.25×10 ^-5 -5.08×10 ^-4 mol/L.

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°C.

Using the above pH clock system, the detectable concentration range of vitamin K3 is the optimal concentration range determined through experiments. In this concentration range, the induction time has a good response to the change of vitamin K3 concentration, and the linear correlation coefficient is large. In addition, the concentration range of each component in the detection solution (pH clock system) is shown in Table 1, and the optimal concentration of the detection solution (pH clock system) obtained through multiple experiments is shown in Table 2:

Table 1: Concentrations of components in the pH clock system

HCHO (mol/L) NaHSO ₃ (mol/L) Na ₂ SO ₃ (mol/L) 0.043-0.0635 0.035-0.0625 0.0035-0.00625

Table 2: Optimum concentrations of components in the pH clock system

HCHO (mol/L) NaHSO ₃ (mol/L) Na ₂ SO ₃ (mol/L) 0.0501 0.0499 0.00499

The specific experimental steps are as follows:

1. Prepare 40mL detection solution (pH clock system) according to the concentration range specified in Table 1, and its temperature is controlled at a specific temperature value between 10-18°C to keep constant; the prepared working electrode (pH composite Electrode, Lei Magnetic, E-331) is inserted into the solution, and the other end of the working electrode is connected to the computer through the potential/temperature/pH comprehensive tester (Jiaxing Disheng Electronic Technology Co., Ltd., ZHFX-595), and the chemical signal acquisition and analysis in the computer is turned on. After the program sets the acquisition time and sampling speed, quickly click the start button to monitor the pH of the solution. The computer records the curve of the collected pH changing with time, that is, the pH clock map. When a substance needs to be detected, the substance to be detected is quickly added at the same time as the reaction of the pH clock system starts, and the pH clock spectrum of pH changing with time is recorded in the same way.

The basic parameters of the pH clock map include:

Induction time: the time required from the start of the pH clock system reaction to the pH jump.

pH jump range: the pH corresponding to the start of the pH jump to the pH corresponding to the end of the pH jump.

Establishment of the working curve for the relationship between the concentration of vitamin K3 in the detection solution and the pH induction time

Use ethanol as a solvent to prepare a vitamin K3 solution with a concentration of 0.0725 mol/L to 0.508 mol/L as a sample solution. When the reaction of the pH clock system starts, add 40 μL of the A series of sample solutions with different concentrations made the concentration of vitamin K3 in the system between 7.25×10 ^-5 mol/L and 5.08×10 ^-4 mol/L; the variation of the response of the pH clock system was the induction time, recorded as t; when When the concentration of vitamin K3 in the system is different, the induction time t of the pH clock system is also different; take the concentration of vitamin K3 in the system as the abscissa and t as the ordinate to draw a graph; when the concentration of vitamin K3 in the system is 7.25×10 ^-5 mol/ When L to 5.08×10 ^-4 mol/L, the pH clock system induction time t has a linear relationship with the concentration of vitamin K3, and the working curve is obtained.

Quantitative detection of vitamin K3

Add a sample to be tested with an unknown concentration into the pH clock system of the detection solution at the beginning of the pH clock system reaction, and the corresponding induction time (t) of the pH clock system can be measured. According to the relationship between t on the working curve and the unknown vitamin K3 The corresponding relationship between concentrations can obtain the concentration of vitamin K3 in the detection system, and then calculate the concentration of vitamin K3 in the test sample.

Description of drawings

Fig. 1 is a graph showing the pH value of the detection solution (pH clock system) changing with time in Example 1 when no sample to be detected is added.

Fig. 2 is a chromatogram of the pH value of the detection solution (pH clock system) changing with time after adding 7.25×10 ^-5 mol/L vitamin K3 in Example 1.

Fig. 3 is a chromatogram of the pH value of the detection solution (pH clock system) changing with time after adding 1.45×10 ^-4 mol/L vitamin K3 in Example 1.

Fig. 4 is the working curve between pH induction time t and vitamin K3 concentration in Example 1.

Fig. 5 is a graph of the pH value of the detection solution (pH clock system) changing with time in Example 2 when no sample to be detected is added.

Fig. 6 is a chromatogram of the pH value of the detection solution (pH clock system) changing with time after adding 2.9×10 ^-4 mol/L vitamin K3 in Example 2.

Fig. 7 is a graph showing the change of pH value of the detection solution (pH clock system) with time after adding 4.35×10 ^-4 mol/L vitamin K3 in Example 2.

Fig. 8 is a working curve between pH induction time t and vitamin K3 concentration in Example 2.

Fig. 9 is a graph showing the pH value of the detection solution (pH clock system) changing with time in Example 3 when no sample to be detected is added.

Fig. 10 is a chromatogram of the pH value of the detection solution (pH clock system) changing with time after adding 4.35×10 ^-4 mol/L vitamin K3 in Example 3.

Fig. 11 is a graph showing the pH value of the detection solution (pH clock system) changing with time after adding 5.08×10 ^-4 mol/L vitamin K3 in Example 3.

Fig. 12 is a working curve between pH induction time t and vitamin K3 concentration in Example 3.

Implementation

Example

The pH clock system with "HCHO-NaHSO ₃ -Na ₂ SO ₃ " as the substrate was used as the detection solution for quantitative analysis of vitamin K3. Add equal volumes of vitamin K3 sample solutions of different concentrations to the pH clock system, and establish a working curve (such as a linear relationship) between the concentration of vitamin K3 in the detection system and the induction time to achieve the purpose of detecting vitamin K3 in the pH clock system. Then calculate the concentration of vitamin K3 in the test sample.

(1) Preparation of detection solution

First, distilled water was used to prepare mixed solutions of 0.12 mol/L HCHO solution, 0.15 mol/L NaHSO ₃ and 0.015 mol/L Na ₂ SO ₃ . Add 10.0mL of distilled aqueous solution, 13.3mL _of NaHSO ₃ -Na ₂ SO _{3 mixed} solution, and 16.7mL of 0.12mol/L HCHO solution to a 50mL small beaker in order to ensure that _each The concentrations of the components were HCHO 0.0501 mol/L, NaHSO ₃ 0.0499 mol/L, and Na ₂ SO ₃ 0.00499 mol/L, the total volume was 40 mL, and the temperature was controlled at 12°C.

At the same time, a series of vitamin K3 sample solutions with different concentrations were prepared using ethanol as a solvent.

(2) Obtain pH clock map

The chromatogram of the pH value of the prepared detection solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (no detection sample is added). As shown in Figure 1. The pH induction time was 230s as a blank control. Another two groups of detection solutions with the same concentration of each component as the above-mentioned detection solutions were prepared. For one of the groups, at the beginning of the reaction, 40 μL of 0.0725 mol/L vitamin K3 sample solution was added to the 40 mL pH clock system, so that the concentration of vitamin K3 in the detection solution was 7.25×10 ^-5 mol/L, The addition of vitamin K3 shortened the induction time to 206s, as shown in Figure 2; for the other group, at the same time as the reaction started, 40 μL of 0.145 mol/L vitamin K3 sample solution was added to the 40 mL pH clock system, so that vitamin K3 The concentration in the detection solution was 1.45×10 ^-4 mol/L, and the addition of vitamin K3 made the induction time 187s, as shown in Figure 3 . Figure 2 and Figure 3 confirm that the different concentrations of vitamin K3 in the detection solution lead to different induction times of the pH clock system. When the concentration of vitamin K3 in the detection system is between 7.25×10 ^-5 mol/L and 5.08×10 ^-4 mol/L, the results of different induction times of the pH clock system due to different concentrations can be observed.

(3) Quantitative detection

Establish a working curve according to the relationship between the concentration of vitamin K3 in the detection system and the induction time, as shown in Figure 4, wherein the abscissa is the concentration of vitamin K3 in the pH clock system, and the ordinate is the induction time t, when the detection system When the concentration of vitamin K3 is between 7.25×10 ^-5 mol/L and 5.08×10 ^-4 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. Accordingly, the quantitative detection of vitamin K3 in the sample can be realized.

Example

(1) Preparation of detection solution

First, distilled water was used to prepare mixed solutions of 0.12 mol/L HCHO solution, 0.15 mol/L NaHSO ₃ and 0.015 mol/L Na ₂ SO ₃ . Add 10mL of distilled aqueous solution, 14mL of NaHSO ₃ -Na ₂ SO ₃ mixed solution, and 16mL of 0.12mol/L HCHO solution to a 50mL small beaker in order to ensure the balance of each component in the "HCHO-NaHSO ₃ -Na ₂ SO ₃ "pH clock system. The concentration is HCHO 0.048mol/L, NaHSO ₃ 0.0525mol/L, Na ₂ SO ₃ 0.00525mol/L, the total volume is 40mL, and the temperature is controlled at 12°C.

At the same time, a series of vitamin K3 sample solutions with different concentrations were prepared using ethanol as a solvent.

(2) Obtain pH clock map

The time-varying spectrum of the pH value of the prepared detection solution was recorded by a computer equipped with a chemical signal acquisition and analysis program (no detection sample was added), as shown in FIG. 5 . The pH induction time was 231s as a blank control. Another two groups of detection solutions with the same concentration of each component as the above-mentioned detection solutions were prepared. For one of the groups, when the reaction started, 40 μL of 0.29 mol/L vitamin K3 sample solution was added to the 40 mL pH clock system, so that the concentration of vitamin K3 in the detection solution was 2.9×10 ^-4 mol/L, The addition of vitamin K3 shortened the induction time to 151s, as shown in Figure 6; for the other group, at the same time as the reaction started, 40 μL of 0.435 mol/L vitamin K3 sample solution was added to the 40 mL pH clock system, so that vitamin K3 The concentration in the detection solution was 4.35×10 ^-4 mol/L, and the addition of vitamin K3 made the induction time 113s, as shown in Figure 7 . Figure 6 and Figure 7 confirm that the different concentrations of vitamin K3 in the detection solution lead to different induction times of the pH clock system. When the concentration of vitamin K3 in the detection system is between 7.25×10 ^-5 mol/L and 5.08×10 ^-4 mol/L, the results of different induction times of the pH clock system due to different concentrations can be observed.

(3) Quantitative detection

Establish a working curve according to the relationship between the concentration of vitamin K3 in the detection system and the induction time, as shown in Figure 8, wherein the abscissa is the concentration of vitamin K3 in the pH clock system, and the ordinate is the induction time t, when in the detection system When the concentration of vitamin K3 is between 7.25×10 ^-5 mol/L and 5.08×10 ^-4 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. Accordingly, the quantitative detection of vitamin K3 in the sample can be realized.

Example

(1) Preparation of detection solution

First, distilled water was used to prepare mixed solutions of 0.12 mol/L HCHO solution, 0.15 mol/L NaHSO ₃ and 0.015 mol/L Na ₂ SO ₃ . Add 10.2mL of distilled aqueous solution, 13.2mL of NaHSO ₃ -Na ₂ SO ₃ mixed solution, and 16.6mL of 0.12mol/L HCHO solution to a 50mL small beaker in order to ensure that each pH value in the "HCHO- NaHSO ₃ - Na ₂ SO ₃ " pH clock system The concentration of the components is HCHO 0.0498 mol/L, NaHSO ₃ 0.0495 mol/L, Na ₂ SO ₃ 0.00495 mol/L, the total volume is 40 mL, and the temperature is controlled at 12°C.

At the same time, a series of vitamin K3 sample solutions with different concentrations were prepared using ethanol as a solvent.

(2) Obtain pH clock map

The chromatogram of the pH value of the prepared detection solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (no detection sample is added). As shown in Figure 9. The pH induction time was 230.6s as a blank control. Another two groups of detection solutions with the same concentration of each component as the above-mentioned detection solutions were prepared. For one of the groups, at the beginning of the reaction, 40 μL of 0.435 mol/L vitamin K3 sample solution was added to the 40 mL pH clock system, so that the concentration of vitamin K3 in the detection solution was 4.35×10 ^-4 mol/L, The addition of vitamin K3 shortened the induction time to 113s, as shown in Figure 10; for the other group, at the same time as the reaction started, 40 μL of 0.508mol/L vitamin K3 sample solution was added to the 40 mL pH clock system, so that vitamin K3 The concentration in the detection solution was 5.08×10 ^-4 mol/L, and the added vitamin K3 made the induction time 102s, as shown in Figure 11 . Figure 10 and Figure 11 confirm that the different concentrations of vitamin K3 in the detection solution lead to different induction times of the pH clock system. When the concentration of vitamin K3 in the detection system is between 7.25×10 ^-5 mol/L and 5.08× ^{10 -4 mol} /L, different concentrations can lead to different induction times of the pH clock system can be observed.

(3) Quantitative detection

Establish a working curve according to the relationship between the concentration of vitamin K3 in the detection system and the induction time, as shown in Figure 12, wherein the abscissa is the concentration of vitamin K3 in the pH clock system, and the ordinate is the induction time t, when in the detection system When the concentration of vitamin K3 is between 7.25×10 ^-5 mol/L and 5.08×10 ^-4 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. Accordingly, the quantitative detection of vitamin K3 in the sample can be realized.

Claims (4)

1. A quantitative detection method of vitamin K3 is characterized in that:

ethanol is used as a solvent to prepare a solution of a sample to be detected;

application of HCHO-NaHSO ₃ - Na ₂ SO ₃ The pH clock reaction system is used as a detection solution, and a map of pH change along with time is recorded; the temperature of the pH clock system is controlled at any specific temperature within the range of 10-18 ℃, when the pH clock reaction starts, the equal volumes of a series of sample solutions to be detected with different concentrations are respectively added into the pH clock system, and quantitative detection of the samples to be detected is realized according to different induction time generated by the system when the concentrations of the solutions to be detected in the pH clock system are different;

detecting the molar concentration of each component in the solutionThe range is as follows: HCHO 0.043-0.0635mol/L, naHSO ₃ 0.035-0.0625mol/L、Na ₂ SO ₃ 0.0035-0.00625mol/L；

The sample to be detected is vitamin K3 solution.

2. The quantitative detection method according to claim 1, wherein: establishing a working curve according to the relation between the concentration of the solution to be detected in the pH clock system and the induction time; wherein the abscissa is the concentration of vitamin K3 in the solution to be detected in the pH clock system, and the ordinate is the induction time t; when the concentration of vitamin K3 in the system is 7.25X10 ^-5 mol/L to 5.08X10 ^-4 When the mol/L ratio is between the two, the induction time t and the concentration of the vitamin K3 form a linear relation, so that the quantitative detection of the vitamin K3 in the sample is realized.

3. The quantitative detection method according to claim 1 or 2, characterized in that: the molar concentration of each component in the detection solution is HCHO 0.0501mol/L, naHSO ₃ 0.0499mol/L、Na ₂ SO ₃ 0.00499mol/L。

4. The quantitative detection method according to claim 1 or 2, characterized in that: the temperature of the pH clock system was controlled at 12℃when the vitamin K3 solution was tested.