A method for detecting histamine content in tylosin tartrate by HPLC

Abstract

The application provides a HPLC detection method for the content of histamine in tylosin tartrate, the content of histamine in a to-be-detected substance is obtained by detecting a test sample solution containing tylosin tartrate raw materials through high performance liquid chromatography, wherein the conditions for the high performance liquid chromatography detection include that a mobile phase includes mobile phase A and mobile phase B, 0.15%-0.2% ammonium acetate solution is used as the mobile phase A, acetonitrile is used as the mobile phase B liquid, and gradient elution is carried out. The application optimizes the chromatographic conditions in the high performance liquid chromatography, establishes the detection method for the content of histamine in tylosin tartrate medicine, and the methodological validation test researches show that the established method has high accuracy, can realize rapid and accurate quantification, and thus the product quality control is achieved.

Description

Technical Field

[0001] This invention belongs to the field of drug detection and testing technology, and in particular relates to an HPLC method for detecting histamine content in tylosin tartrate. Background Technology

[0002] Tylosin tartrate is a macrolide antibiotic, specifically designed to combat mycoplasma, Gram-positive bacteria, and some Gram-negative bacteria. It boasts a broad antibacterial spectrum and low toxicity. As a widely used veterinary antibiotic internationally, tylosin tartrate is commonly added to livestock and poultry feed. Its main indications are diarrhea and respiratory diseases caused by susceptible bacteria and mycoplasma. Adding tylosin to feed can significantly promote growth rate and shorten the feeding cycle.

[0003] Histamine is a biogenic amine found in many plants and animals. Small amounts of histamine play an important regulatory role in responses and inflammation, but excessive amounts can lead to histamine poisoning. High concentrations of histamine are often found in fermented products. Most veterinary drugs are processed through fermentation, but there are currently no regulations specifying the limits for histamine in veterinary drugs. Organic impurities in these drugs can cause significant interference, and a mature and efficient method for measuring histamine content in veterinary drugs is lacking. Since conventional spectroscopic methods are easily affected by impurities, this paper uses high-performance liquid chromatography (HPLC) to determine the histamine content in tylosin tartrate. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting histamine content in tylosin tartrate. The method uses high performance liquid chromatography to detect histamine content in tylosin tartrate. By optimizing the process steps and gradient elution conditions, the separation degree can be improved and the analysis time can be shortened.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] An HPLC method for detecting histamine content in tylosin tartrate includes:

[0007] The test solution containing tylosin tartrate was analyzed by high-performance liquid chromatography (HPLC) to determine the histamine content in the analyte. The HPLC conditions were as follows:

[0008] The chromatographic column is a C18 column;

[0009] The mobile phases are: Mobile phase A: 0.15%-0.2% ammonium acetate solution, Mobile phase B: acetonitrile;

[0010] The elution method is gradient elution.

[0011] In the gradient elution process, the volume ratio of mobile phase A to mobile phase B is:

[0012]

[0013]

[0014] The detection wavelength of the high-performance liquid chromatography method is 254 nm.

[0015] The column temperature for the high-performance liquid chromatography method is 35°C.

[0016] The flow rate of the high-performance liquid chromatography method is 1.0 mL / min.

[0017] The method for preparing the test solution of the tylosin tartrate raw material includes:

[0018] ① Weigh 0.4g of tylosin, add 5mL of water and stir to dissolve. At the same time, slowly add an equal volume of 10% trichloroacetic acid solution. After filtration, add 5mL of sodium hydroxide and 0.3mL of benzoyl chloride. Place in a constant temperature shaker and mix well. Then add 2.5g of sodium chloride and continue to shake to dissolve, obtaining the solution.

[0019] ② Transfer the solution collected in step ① into a separatory funnel, add diethyl ether for two extractions, allow to stand and separate the layers, and then combine the extracts to obtain the final extract.

[0020] ③ Evaporate the extract obtained in step ② to dryness in a water bath, then add 2 mL of methanol to completely dissolve it, thus obtaining the test solution.

[0021] The concentration of the sodium hydroxide solution mentioned in step ① is 2-3 mol / L.

[0022] The constant temperature oscillator in step ① operates under the following conditions: oscillation time 20-30 min, temperature 37℃, rotation speed 200 rpm. When the oscillation reaches the middle of the time, 2.5 g of sodium chloride is added and oscillation continues to dissolve.

[0023] In step ①, the extraction reagent is diethyl ether, and the extraction is performed by shaking twice, 5 mL each time, for 3 min.

[0024] In step ①, the temperature for removing the solvent from the extract is 50-60℃.

[0025] This invention has the following beneficial results:

[0026] This invention, through experimental refinement, establishes a method for detecting histamine in tylosin tartrate. The device precision RSD is 0.13%; sample stability is 1.25%; repeatability RSD is 2.34%; recovery RSD is 1.56%; and the limit of detection (LOD) and limit of quantitation (LOQ) are 0.06% and 0.81%, respectively. This indicates that the extraction and detection method has high accuracy, strong specificity, good reproducibility, and high sample recovery. It can efficiently and accurately detect the histamine content in tylosin tartrate, enabling effective control of product quality. Attached Figure Description

[0027] Figure 1 The detection chromatogram of the first batch of reference standards in Example 1;

[0028] Figure 2 The detection chromatogram 1 is the first batch of test samples in Example 1;

[0029] Figure 3 The detection chromatogram 2 is the first batch of test samples in Example 1;

[0030] Figure 4 The detection chromatogram of the second batch of reference standards in Example 1;

[0031] Figure 5 The detection chromatogram of the second batch of test samples in Example 1;

[0032] Figure 6 The detection spectrum of the non-injected sample in Example 1;

[0033] Figure 7 This is the standard curve for Example 1. Detailed Implementation

[0034] Specific Implementation Method 1: This implementation method provides a method for detecting histamine content in tylosin tartrate, which includes the following steps:

[0035] (1) Preparation of test solution:

[0036] ① Weigh 0.4g of tylosin, add 5mL of water to dissolve, and add the same volume of 10% trichloroacetic acid solution while shaking. After filtration, add 5mL of 2-3mol / L sodium hydroxide and 0.3mL of benzoyl chloride. Place in a constant temperature shaker and shake for 20-30min at 37℃ and 200r / min. When shaking reaches the middle of the time, add 2.5g of sodium chloride and continue shaking to dissolve.

[0037] ② Transfer the solution collected in the previous step into a separatory funnel, extract it twice with ether for 3 minutes each time, and let it stand to separate into layers.

[0038] ③ Evaporate the extract obtained in step ② to dryness in a water bath at 50-60℃, add 2mL of methanol to dissolve the residue, mix well, and use as the test solution.

[0039] (2) Preparation of histamine standard solution:

[0040] Prepare 2.5 mL of standard stock solutions of 0.0008 mg / mL, 0.004 mg / mL, 0.008 mg / mL, 0.012 mg / mL, and 0.016 mg / mL respectively (weigh 8-10 mg of histamine standard, dissolve in water, dilute to 100 mL, and shake well). Then add the same volume of 10% trichloroacetic acid solution to each stock solution and repeat steps ①②③ above to obtain the standard solutions.

[0041] (3) Blank solution:

[0042] Take 2.5 mL of water, add the same volume of 10% trichloroacetic acid solution, and repeat steps ①②③ above to obtain a blank solution.

[0043] (4) Measurement:

[0044] Measure 10 μL each of the blank solution, standard solution, and test solution, and inject them into the HPLC system. Record the chromatograms. Plot a linear graph with histamine peak area on the ordinate and concentration on the abscissa. Calculate the histamine content in the test sample based on the peak area using the standard curve method.

[0045] Chromatographic conditions: Column: C18 column, size: 4.6*150mm, particle size: 5μm; flow rate: 1.0mL / min; wavelength: 254nm; column temperature: 35℃; injection volume: 10μL; gradient elution was performed (mobile phase A: 0.15%-0.2% ammonium acetate solution; mobile phase B: acetonitrile).

[0046] Specific Implementation Method 2: This implementation method differs from Specific Implementation Method 1 in that the concentration of the sodium hydroxide solution is 2 mol / L. Everything else is the same as in Specific Implementation Method 1.

[0047] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 1 in that the oscillation time of the constant temperature oscillator is 20 minutes. Everything else is the same as in Specific Implementation Method 1.

[0048] Specific Implementation Method 4: This implementation method differs from Specific Implementation Method 1 in that the water bath evaporation temperature is 50℃. Everything else is the same as in Specific Implementation Method 1.

[0049] Specific Implementation Method 5: This implementation method differs from Specific Implementation Method 1 in that the ammonium acetate content in the preparation of mobile phase A is 0.15%. Everything else is the same as in Specific Implementation Method 1.

[0050] Specific Implementation Method 6: This implementation method differs from Specific Implementation Method 1 in that the gradient elution time is 0-35 minutes. Everything else is the same as in Specific Implementation Method 1.

[0051] The scope of this invention is not limited to the above-described embodiments; a combination of one or more specific embodiments can also achieve the purpose of the invention.

[0052] Example 1

[0053] The HPLC determination of histamine content in tylosin tartrate is performed according to the following steps:

[0054] Chromatographic conditions

[0055] Chromatographic column: C18 column, dimensions: 4.6*150mm, particle size: 5μm; flow rate: 1.0mL / min, wavelength: 254nm, column temperature: 35℃, injection volume: 10μL, with gradient elution.

[0056]

[0057] ① Preparation of the test sample: Weigh 0.4 g of tylosin, dissolve it in 5 mL of water, and add an equal volume of 10% trichloroacetic acid while shaking. After filtration, add 5 mL of 2 mol / L sodium hydroxide solution and 0.3 mL of benzoyl chloride. Place the solution in a constant temperature shaker and shake at 37°C for 10 min. Then add 2.5 g of sodium chloride and continue shaking for 10 min to dissolve it. Transfer the dissolved solution to a separatory funnel and extract it twice with diethyl ether, 3 min each time. Evaporate the extracted solution to dryness in a 50°C water bath, add 2 mL of methanol to dissolve and mix the residue to obtain the test sample solution.

[0058] ② Preparation of histamine standard solutions: Prepare 2.5 mL of standard stock solutions with concentrations of 0.0008 mg / mL, 0.004 mg / mL, 0.008 mg / mL, 0.012 mg / mL, and 0.016 mg / mL, respectively. Then add the same volume of 10% trichloroacetic acid solution to each stock solution. Repeat the subsequent steps of the test solution preparation to obtain the standard solutions.

[0059] ③ Blank solution: Take 2.5 mL of water, add the same volume of 10% trichloroacetic acid solution, and repeat the subsequent steps of the test sample preparation to obtain the blank solution.

[0060] ④ Determination: Measure 10 μL each of the blank solution, standard solution, and test solution, inject them into the HPLC system, and record the chromatograms. Plot a linear graph with histamine peak area on the ordinate and concentration on the abscissa. Calculate the histamine content in the test sample based on the peak area using the standard curve method.

[0061] This embodiment discloses a method for detecting histamine in tylosin tartrate. During the experiment, an internal standard method was used to plot a standard curve for histamine content. Currently, there is no HPLC method for detecting histamine in tylosin tartrate, and other methods use external standard methods to plot standard curves. Compared to these, the internal standard method is more accurate and reliable. The selection of extraction reagents, extraction methods, and HPLC detection conditions were determined to ensure good HPLC resolution and effective detection of histamine content in tylosin tartrate.

[0062] The results show that the standard curve is Y = 5001.1x + 7.7912, with good linearity and R0. 2 =0.9992. A certain concentration of histamine standard was added to tylosin tartrate, and the sample was detected according to the established HPLC method. The same sample was injected five times consecutively, and the RSD of the HPLC precision was 0.13%. The same sample was injected at 0, 2, 4, 6, 12, and 24 hours, and the RSD of the sample stability was 1.25%. The experiment proved that the method established in this example has high accuracy, strong specificity, and good reproducibility, and can effectively detect the histamine content in tylosin tartrate.

[0063] Method Validation

[0064] The prepared histamine standard was diluted 10, 20, 50, 80, 100 and 200 times respectively, and then added to the test sample of this experiment. The test was performed using the detection method of this experiment. The experiment was repeated 3 times to obtain the recovery rate and standard deviation. The histamine content in the sample was calculated according to formula (I).

[0065]

[0066] In the formula: X — the histamine content in tylosin tartrate, in units of (μg / g);

[0067] C—The concentration of histamine in tylosin in the sample solution is (detection peak area - 7.7912) / 5001.1 * 0.04, in units of (μg / μL);

[0068] m — the mass of the sample, in grams (g);

[0069] V – Volume of the sample solution, in milliliters (mL);

[0070] When the concentration of the standard solution added to the sample solution was between 0.0008 and 0.016 mg / mL, the average recovery rate of histamine in tylosin tartrate ranged from 97.89% to 101.24%. The limit of detection (LOD) was 0.04 μg / mL using a standard solution concentration with a signal-to-noise ratio (S / N) of 3:1 as the standard concentration, and the limit of quantitation (LOQ) was 0.77 μg / mL using a standard solution concentration with a signal-to-noise ratio (S / N) of 10:1 as the standard concentration.

[0071] The results of testing three batches of samples using the methods determined above are as follows:

[0072] Detection results of histamine in the test sample

[0073]

Claims

1. An HPLC method for detecting histamine content in tylosin tartrate, comprising: The test solution containing tylosin tartrate was analyzed by high-performance liquid chromatography (HPLC) to determine the histamine content in the analyte. The HPLC conditions were as follows: The chromatographic column is a C18 column; The mobile phases are: Mobile phase A: 0.15%-0.2% ammonium acetate solution, Mobile phase B: acetonitrile; The elution method is gradient elution.

2. The detection method according to claim 1, characterized in that... In the gradient elution process, the volume ratio of mobile phase A to mobile phase B is:

3. The detection method according to claim 1, characterized in that... The detection wavelength of the high-performance liquid chromatography method is 254 nm.

4. The detection method according to claim 1, characterized in that... The column temperature for the high-performance liquid chromatography method is 35°C.

5. The detection method according to claim 1, characterized in that... The flow rate of the high-performance liquid chromatography method is 1.0 mL / min.

6. The detection method according to claim 1, characterized in that... The method for preparing the test solution of the tylosin tartrate raw material includes: ① Weigh 0.4g of tylosin, add 5mL of water and stir to dissolve. At the same time, slowly add an equal volume of 10% trichloroacetic acid solution. After filtration, add 5mL of sodium hydroxide and 0.3mL of benzoyl chloride. Place in a constant temperature shaker and mix well. Then add 2.5g of sodium chloride and continue to shake to dissolve, obtaining the solution. ② Transfer the solution collected in step ① into a separatory funnel, add diethyl ether for two extractions, allow to stand and separate the layers, and then combine the extracts to obtain the final extract. ③ Evaporate the extract obtained in step ② to dryness in a water bath, then add 2 mL of methanol to completely dissolve it, thus obtaining the test solution.

7. The detection method according to claim 6, characterized in that... The concentration of the sodium hydroxide solution mentioned in step ① is 2-3 mol / L.

8. The detection method according to claim 6, characterized in that... The operating conditions of the constant temperature oscillator in step ① are as follows: oscillation time 20-30 min, temperature 37℃, rotation speed 200 rpm. When the oscillation reaches the middle of the time, add 2.5 g of sodium chloride and continue oscillating to dissolve.

9. The detection method according to claim 6, characterized in that... The extraction reagent in step ① is diethyl ether. The extraction is performed by shaking twice, 5 mL each time, for 3 min each time.

10. The detection method according to claim 6, characterized in that... The temperature for removing the solvent from the extract in step ① is 50-60℃.

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