High performance liquid chromatography for simultaneously detecting isatin, indigo blue and indirubin

Abstract

The invention relates to a high performance liquid chromatography for simultaneously detecting isatin, indigo blue and indirubin. Chromatographic conditions are as follows: a chromatographic column is an Agilent ZORBAX Eclipse XDB-C18 column with the column length of 150mm, the inner diameter of 4.6 mm and a filler of 3.5 microns; the column temperature is 30 DEG C, the VWD detection wavelength is 300 nm, the flow velocity is 0.8 mL / min, the sample size is 5 [mu] L, and the mobile phases are as follows: the phase A is pure water, and the phase B is acetonitrile. The method not only can accurately judge whether indigo in the product comes from plants or is industrially synthesized, but also can deeply study the degradation degree and shelf life of indigo, and provides a scientific basis for the quality and application of indigo dye.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a high performance liquid chromatography method for simultaneously detecting indigo carmine, indigo blue and indirubin. Background Art

[0002] Indigo is a vat dye with a history of more than 3,000 years and is one of the oldest pigments known to mankind. Indigo dye is widely used in the textile and food industries due to its excellent dyeing effect. Indigo also has excellent anti-inflammatory and antioxidant effects and significant antibacterial and antiviral activity, which makes it widely used in the pharmaceutical industry.

[0003] Indigo can be divided into plant indigo, biological indigo and industrial indigo according to its source. Among them, industrial indigo is obtained by chemical synthesis. Compared with plant indigo, it has the characteristic of high yield. However, it is far inferior to plant indigo in terms of environmental protection. Biological indigo has the characteristics of high yield and environmental protection, and has significant advantages in environmental protection, health and sustainable development.

[0004] Indirubin is a purple-red substance, a diindole alkaloid isolated from plant indigo. It is an isomer of indigo and has good anti-inflammatory and antioxidant functions. It has an inhibitory effect on a variety of transplanted animal tumors and can destroy leukemia cells. Indirubin can also be used as a detection substance for the naturalness of plant indigo. Its presence is a green natural sign. Since both plant indigo and biological indigo contain indirubin, while industrial indigo does not contain indirubin, it can be used as a marker to distinguish natural indigo (plant indigo and biological indigo) from industrial indigo.

[0005] Isatin is an orange-red indole derivative that can be obtained by oxidation of indigo. Studies have shown that ultraviolet light with strong energy has a greater impact on the degradation of indigo. In the photodecomposition process, photooxidation plays a dominant role in the photodegradation process, and the fading of the dye is mainly driven by oxidants. Data show that the main degradation product of industrial indigo and plant indigo is isatin. Therefore, the content of isatin can be used as the main indicator for evaluating the degradation of indigo.

[0006] As a natural dye with a long history, the identification of indigo’s source and quality has always been the focus of the industry. However, the current research on the source and degradation degree of indigo can only be tested and evaluated separately, and there is no analytical method that can simultaneously detect indigo, indirubin, and indigo carmine. Therefore, the development of an HPLC method for the simultaneous detection of indigo, indigo, and indirubin is crucial to accurately determine whether the indigo in the product is derived from plants or organisms, or is chemically synthesized, and to evaluate its degradation degree. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects in the prior art and provide a high performance liquid chromatography method for simultaneously detecting indigo red, indigo and indirubin, which can not only accurately determine whether the indigo in the product is derived from nature (plant or biological) or industrial synthesis, but also deeply study its degradation degree, providing a scientific basis for the quality and application of indigo dyes.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] A high performance liquid chromatography method for simultaneously detecting indigo, indigo and indirubin, the chromatographic conditions are as follows:

[0010] Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 column with a length of 150 mm, an inner diameter of 4.6 mm, and a filler of 3.5 μm; column temperature 30°C, VWD detection wavelength 300 nm, flow rate 0.8 mL / min, injection volume 5 μL, mobile phase: phase A is pure water, phase B is acetonitrile.

[0011] As a further technical solution, the mobile phase is run using a gradient elution program.

[0012] As a further technical solution, the gradient elution procedure is as follows:

[0013] Time (min) Phase A (volume percentage) % Phase B (volume percentage) % 0 70 30 5 70 30 14 50 50 15 50 50 25 70 30 30 70 30 .

[0014] As a further technical solution, the standard solutions of indigo, indigo and indirubin and the indigo sample solution are prepared using DMSO containing 0.5% 2,6-di-tert-butyl-p-cresol (BHT) as a solvent.

[0015] As a further technical solution, when preparing the standard solutions of indigo, indigo and indirubin and the indigo sample solution, they are ultrasonicated for 10 minutes in an ultrasonicator at a frequency of 40 Hz to promote dissolution.

[0016] As a further technical solution, it is used to detect the content of indigo, indigo and indirubin in biological indigo.

[0017] As a further technical solution, the indigo solution is irradiated with ultraviolet light at a rated power of 50 Hz for 1 hour to cause photodegradation to produce indigo red.

[0018] As a further technical solution, the wavelength of the ultraviolet light is 253.7 nm.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention selects the solvent and adjusts the mobile phase, which not only enables the effective separation of indigo, indirubin and indigo carmine, realizes the simultaneous detection of the three, improves the detection efficiency, but also enables the effective separation of indigo carmine and the solvent peak, thereby realizing the accurate measurement of indigo carmine and providing a methodological basis for the degradation evaluation of indigo.

[0021] (2) The high performance liquid chromatography method for the simultaneous detection of indigo, indigotin and indigo carmine developed in the present invention can accurately determine whether the indigo dye is derived from plants or organisms or is chemically synthesized, which is crucial for ensuring the quality and traceability of dyed products; in addition, it can also deeply analyze the degradation degree of indigo dye and guide production and storage by detecting the degradation degree of indigo dye.

[0022] (3) Compared with traditional detection methods, the high performance liquid chromatography method of the present invention can complete the detection more quickly and accurately, greatly improving the detection efficiency; it provides a solid scientific basis for scientifically evaluating the quality and application of indigo dyes, and for product quality control and technological innovation in the dyeing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the state in which isatin, indigo and indirubin are dissolved in DMSO.

[0024] exist Figure 1 In the middle, A: indigo, B: indigo, C: indigotine;

[0025] Figure 2 These are the HPLC chromatograms of indigo, indigo blue and indirubin standards.

[0026] exist Figure 2 In the figure, A: indigo standard, B: indigo standard, C: indirubin standard;

[0027] Figure 3 This is a high performance liquid chromatogram of a mixed standard sample containing indigo red, indigo blue and indirubin.

[0028] Figure 4 This is a comparison chart of HPLC before and after indigo degradation by ultraviolet light;

[0029] exist Figure 4 In the figure, A: biological indigo sample; B: biological indigo photolysis sample. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0032] Biological indigo: isolated and purified from the fermentation broth of indigo-producing engineered bacteria.

[0033] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0034] Example 1

[0035] A high performance liquid chromatography detection method for simultaneously detecting indigo, indigo and indirubin, the steps are as follows:

[0036] Step 1: Preparation of standard solutions of indigo, indigo and indirubin

[0037] (1) Preparation of indigo carmine standard solution: weigh 5 mg of indigo carmine standard and dissolve it in 50 mL of DMSO solution (containing 0.5% BHT). Ultrasonicate it in a 40 Hz ultrasonic instrument for 10 min until it is completely dissolved. The concentration is 0.1 g / L. Dilute it 10 times with DMSO. Centrifuge it at 12000 rpm for 10 min. Take 200 μL of it and put it into an inner tube and store it in a brown liquid phase vial. A 10 mg / L indigo carmine standard solution is obtained.

[0038] (2) Preparation of indigo standard solution: weigh 5 mg of indigo standard and dissolve it in 50 mL of DMSO (containing 0.5% BHT) solution. Ultrasonicate it in a 40 Hz ultrasonic instrument for 10 min until it is completely dissolved. The concentration is 0.1 g / L. Dilute it 10 times with DMSO. Centrifuge it at 12000 rpm for 10 min. Take 200 μL and put it into an inner tube and store it in a brown liquid phase vial. A 10 mg / L indigo standard solution is obtained.

[0039] (3) Preparation of indigo carmine standard solution: weigh 5 mg of indigo carmine standard and dissolve it in 50 mL of DMSO solution (containing 0.5% BHT). Ultrasonicate it in a 40 Hz ultrasonic instrument for 10 min until it is completely dissolved, and the concentration is 0.1 g / L. Then dilute it 10 times with DMSO, centrifuge it at 12000 rpm for 10 min, take 200 μL of it and put it into an inner tube and store it in a brown liquid vial to obtain a 10 mg / L indigo carmine standard solution.

[0040] The color state diagram of the above indigo red standard solution, indigo blue standard solution and indirubin standard solution is shown in Figure 1 .

[0041] Step 2: Preparation of mixed standard solution:

[0042] Take 2mL of 0.1g / L indigo standard solution, 1mL of 0.1g / L indigo standard solution and 2mL of 0.1g / L indigo red standard solution respectively, mix them evenly, make up to 10mL, centrifuge at 12000rpm for 10min, take 200μL, put it into an inner tube and store it in a brown liquid phase vial to obtain a mixed standard solution containing indigo, indigo and indigo red with concentrations of 20mg / L, 10mg and 20mg / L respectively.

[0043] Step 3: Preparation of bioindigo photodegradation samples

[0044] Preparation of biological indigo sample solution: Weigh 5 mg of biological indigo and dissolve it in 50 mL of DMSO (containing 0.5% BHT) solution, sonicate it in a 40 Hz ultrasonic instrument for 10 minutes until it is completely dissolved, and the concentration is 0.1 g / L. Then dilute it 10 times with DMSO, centrifuge it at 12000 rpm for 10 minutes, take 200 μL and put it into the inner tube and store it in a brown liquid phase vial. A 10 mg / L biological indigo sample solution is obtained.

[0045] Preparation of biological indigo photodegradation sample solution: Take the biological indigo sample solution, dilute it with DMSO to 8 mg / L, and centrifuge it at 12000 rpm for 10 minutes to obtain the biological indigo sample solution. Place the sample solution in a transparent 10 mL plastic ep tube and irradiate it under ultraviolet light with a wavelength of 253.7 nm and a frequency of 50 Hz for 1 hour. At this time, indigo undergoes photodegradation to produce indigo red, and the biological indigo photodegradation sample solution is obtained.

[0046] Step 4: HPLC detection

[0047] 1. Chromatographic detection conditions

[0048] A ZORBAX Eclipse XDB-C18, 150 mm × 4.6 mm × 3.5 μm column was used;

[0049] Mobile phase A: pure water, B: acetonitrile, gradient elution; the elution gradient is as follows:

[0050] Time (min) Phase A (volume percentage) % Phase B (volume percentage) % 0 70 30 5 70 30 14 50 50 15 50 50 25 70 30 30 70 30

[0051] Mobile phase flow rate: 0.8 mL / min;

[0052] Detection wavelength: 300nm;

[0053] Chromatographic column temperature: 30°C;

[0054] The injection volume was 5 μL;

[0055] Detector: Agilent 1260 Infinity II VWD detector;

[0056] 2. Experimental steps

[0057] (1) Prepare the mobile phase by selecting 1000 mL of pure water that meets the HPLC mobile phase standard and 1000 mL of Aladdin chromatography grade acetonitrile as the mobile phase. Both mobile phases need to be ultrasonicated for 30 min at a frequency of 40 Hz to degas.

[0058] (2) Adjust the HPLC to balance the column pressure and signal;

[0059] (3) The indigo standard solution, indirubin standard solution, indigo carmine standard solution, mixed standard solution, bio-indigo sample solution and bio-indigo photodegradation sample solution were detected by liquid chromatography-ultraviolet detector, and the chromatogram was recorded (see Figure 2-4 ).

[0060] 3. Results Analysis

[0061] Depend on Figure 2 and Figure 3 The analysis shows that the method of the present invention can effectively separate the three substances, namely, indigo, indigo and indirubin. Compared with the traditional technology, the method of the present invention shortens the peak elution time and simplifies the preparation procedure of the mobile phase. At the same time, it effectively separates the DMSO solvent peak and the indigo peak, making the experimental data clearer, more accurate and faster.

[0062] Depend on Figure 4 The analysis shows that after the biological indigo sample is degraded by ultraviolet light, the indigo peak area decreases and the indigo red peak area increases, indicating that the sample has been oxidized and degraded by ultraviolet light. It also contains a certain amount of indigo red. Similarly, this method can detect the degradation degree of chemical indigo and plant indigo.

[0063] Example 2: Quantitative determination of indigo, indigo and indirubin

[0064] The quantitative determination of indigo, indigo and indirubin comprises the following steps:

[0065] Step 1: Draw the standard curve:

[0066] The mixed standard solution in Example 1 was diluted in a gradient manner to obtain standard solutions of different concentrations, and then the chromatographic conditions in Example 1 were used for detection, and the standard curves of indigo, indirubin and isatin were plotted with the concentration as the abscissa and the peak area as the ordinate to obtain the standard curve equation, as follows:

[0067] Indigo carmine standard curve: y = 30.506x + 2.65;

[0068] Indigo standard curve: y = 43.436x + 11.354;

[0069] Indirubin standard curve: y = 45.225x + 1.3913;

[0070] Step 2, the biological indigo sample solution and the biological indigo photodegradation sample solution prepared in Example 1 were detected, and the concentrations of each substance in the biological indigo sample solution and the biological indigo photodegradation sample solution were calculated according to the standard curve equation and the peak area. The results are as follows: in the biological indigo sample solution (sample not subjected to photodegradation), the indigo red concentration was 0.02 mg / L, the indigo concentration was 3.65 mg / L, and the indigo red concentration was 0.08 mg / L; in the biological indigo photodegradation sample solution (sample solution after photodegradation), the indigo red concentration was 2.96 mg / L, the indigo concentration was 0.32 mg / L, and the indigo red concentration was 0.07 mg / L.

[0071] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A high performance liquid chromatography method for simultaneously detecting indigo, indigo and indirubin, characterized in that: The chromatographic conditions were as follows: Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 column with a length of 150 mm, an inner diameter of 4.6 mm, and a filler of 3.5 μm; column temperature 30°C, VWD detection wavelength 300 nm, flow rate 0.8 mL / min, injection volume 5 μL, mobile phase: phase A is pure water, phase B is acetonitrile.

2. The high performance liquid chromatography method for simultaneously detecting indigo, indigo and indirubin according to claim 1, characterized in that: The mobile phase was run using a gradient elution program.

3. The high performance liquid chromatography method for simultaneously detecting indigo, indigo and indirubin according to claim 2, characterized in that: The gradient elution program is as follows: 。 4. The high performance liquid chromatography method for simultaneously detecting indigo, indigo and indirubin according to claim 1, characterized in that: The standard solutions of indigo, indigo and indirubin and the indigo sample solution were prepared using DMSO containing 0.5% 2,6-di-tert-butyl-p-cresol as solvent.

5. The high performance liquid chromatography method for simultaneously detecting indigo, indigo and indirubin according to claim 4, characterized in that: When preparing the standard solutions of indigo, indigo and indirubin and the indigo sample solution, they were ultrasonicated for 10 min in an ultrasonicator at a frequency of 40 Hz to promote dissolution.

6. The high performance liquid chromatography method for simultaneously detecting indigo, indigo and indirubin according to claim 1, characterized in that: Used to detect the content of indigo, indigo and indirubin in biological indigo.

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