Method for determining the content of 25-hydroxycholecalciferol in a feed additive

Abstract

This invention discloses a method for determining the content of 25-hydroxycholecalciferol in feed additives, belonging to the field of additive detection technology. The method includes the following steps: preparing a standard working solution; preparing the sample solution: weighing the sample and placing it in a methanol-water solution, sonicating at 55℃ for 30 min, cooling, and then filtering to obtain the sample solution; liquid chromatography detection: injecting the standard working solution and the sample solution separately for detection, using retention time for qualitative analysis and peak area external standard method for quantitative analysis, to obtain the content of 25-hydroxycholecalciferol in the sample solution. This invention can significantly improve the carrier dissolution effect, reduce target degradation, and improve chromatographic separation effect. The detection stability is significantly better than conventional operations, and it can simultaneously meet the quality detection requirements of both raw material and coated 25-hydroxycholecalciferol feed additives, providing reliable technical support for standardized testing in the industry.

Description

Technical Field

[0001] This invention belongs to the field of additive detection technology, specifically relating to a method for determining the content of 25-hydroxycholecalciferol in feed additives. Background Technology

[0002] 25-Hydroxycholic acid is a commonly used, highly effective functional vitamin additive in the livestock industry. It boasts high bioavailability and can effectively improve animal bone development and immune performance. Currently, domestic testing standards are divided into two sets of industry standards (NY / T4688-2025 and T / CFIAS 3039-2025) for raw materials and coated formulations, both employing high-performance liquid chromatography (HPLC). However, these standards only specify the basic testing framework and lack standardization of key processes such as pretreatment temperature control, extraction time, and cooling methods. Conventional testing often relies on experience, leading to problems such as insufficient dissolution of coated samples, degradation of the target analyte by heat and light, large errors due to solvent evaporation, poor peak shape, inadequate separation, and poor data repeatability. These issues make it difficult to meet the requirements for precise quantification of trace samples. Summary of the Invention

[0003] Technical problem solved: To address the above-mentioned technical problems, this invention provides a method for determining the content of 25-hydroxycholecalciferol in feed additives, which is applicable to both raw materials and coated formulations, with controllable error and strong stability.

[0004] Technical solution: A method for determining the content of 25-hydroxycholecalciferol in feed additives, comprising the following steps: S1. Prepare the standard working solution; S2. Preparation of the test sample solution: Weigh the sample and place it in a methanol aqueous solution, sonicate at 55℃ for 30 min, cool and then filter to obtain the test sample solution; S3. Liquid chromatography detection: The standard working solution and the sample solution to be tested are injected separately for detection. The retention time is used for qualitative analysis, and the peak area external standard method is used for quantitative analysis to obtain the content of 25-hydroxycholecalciferol in the sample solution to be tested.

[0005] Preferably, the standard working solution is prepared by dissolving a 25-hydroxycholecalciferol aqueous compound standard in an aqueous methanol solution.

[0006] Preferably, in step S2, the cooling method is ice-water bath cooling.

[0007] Preferably, steps S1 and S2 are performed entirely in the dark.

[0008] Preferably, in step S2, the volume ratio of methanol to water in the methanol-water solution is 80:20.

[0009] Preferably, in step S2, a 0.22 μm organic filter membrane is used for filtration.

[0010] Preferably, the liquid chromatography conditions in step S3 are as follows: C18 column with dimensions of 4.6 mm × 150 mm and 5 μm; mobile phase of methanol-water solution; flow rate of 1.0 mL / min; column temperature of 26~28 ℃; detection wavelength of 265 nm; and injection volume of 20 μL.

[0011] Furthermore, the volume ratio of methanol to water in the mobile phase is 80:20.

[0012] Furthermore, the mobile phase is pretreated by degassing through a 0.45 μm filter membrane.

[0013] Preferably, after the detection in step S3, the column is maintained by a segmented gradient rinsing: first rinse with a 10% (v / v) methanol aqueous solution for 1 hour, and then rinse with pure methanol for 1 hour.

[0014] Beneficial Effects: This invention employs the industry-standard 80:20 methanol-water system without altering the publicly disclosed ratio, focusing on optimizing the supporting pretreatment process. Traditional methods use only the solvent but lack standardized temperature control, cooling, and light-protection procedures, resulting in incomplete extraction, peak tailing, low impurity separation, and large data fluctuations. This invention, through optimized integrated processes, improves extraction recovery by approximately 4.2% and impurity separation by approximately 15.6% compared to traditional extensive processes. It significantly enhances carrier dissolution, reduces target analyte degradation, improves chromatographic separation, and significantly outperforms conventional operations in detection stability.

[0015] Specifically, this invention: (1) Based on the general solvent system, establish a complete set of processes for temperature-controlled cell disruption, timed extraction and rapid stabilization of coated samples to fill the gap in industry parameters; (2) The first 55 ℃ constant temperature coupled ice water bath cooling process is used to balance high extraction rate and low degradation rate, and solve the pain points of difficult cell wall breaking and easy degradation of coated samples; (3) Achieve light protection, standardized operation and column maintenance throughout the entire process, greatly improving the long-term stability of the system; (4) One process can be adapted to both raw materials and coating products, making it more versatile and more suitable for routine quality inspection by enterprises.

[0016] This invention effectively solves the problems of incomplete extraction, easy degradation of target substances, poor peak shape, and large data fluctuations in traditional methods by optimizing temperature-controlled ultrasonic timed extraction, rapid cooling and shaping, and full-process light protection and chromatographic preservation. The method exhibits excellent linearity (R0). 2 With a strength of 0.9999, high accuracy and precision, and good solution stability, it can simultaneously meet the quality testing requirements of both raw material and coated 25-hydroxycholecalciferol feed additives, and can provide reliable technical support for standardized testing in the industry. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments.

[0018] 1. Experimental Principle The sample was extracted with methanol-water solution under constant temperature and ultrasonication to fully dissolve the free and coated 25-hydroxycholecalciferol. After filtration to remove impurities, the sample was separated using a C18 reversed-phase chromatographic column and detected at 265 nm ultraviolet wavelength. The sample content was determined by retention time for qualitative analysis and external standard method for quantitative analysis by peak area.

[0019] 1.2 Reagents and Materials Chromatographic grade methanol, GB / T 6682 Grade I laboratory water, and 25-hydroxycholecalciferol aqueous standard (CAS: 63283-36-3, purity ≥98.0%) were used to prepare an 80:20 volume ratio methanol-water solution as the extraction and mobile phase solvent.

[0020] 1.3 Preparation of Standard Solutions Standard stock solution (1 mg / mL): Accurately weigh 25 mg (accurate to 0.01 mg) of standard, dissolve in methanol and bring to a final volume of 25 mL in a brown volumetric flask. Store at -18 ℃ protected from light for extended periods; shelf life is 6 months. The experimental working solution should be prepared immediately after dilution using an 80:20 methanol-water system, and handled in the dark throughout the process to minimize degradation losses.

[0021] 1.4 Instruments and Equipment High-performance liquid chromatograph (equipped with a UV detector), analytical balance of 0.0001 g / mL, digitally controlled ultrasonic water bath, ice-water bath device, 0.22 μm and 0.45 μm organic microporous filter membranes, brown light-proof volumetric flasks, etc.

[0022] 1.5 Sample Pretreatment The entire process was conducted in the dark. Accurately weigh 1 g of sample (to a precision of 0.01 g) and place it in a 25 mL brown volumetric flask. Add 20 mL of 80:20 methanol-water solution. An optimized two-stage temperature control process was employed: ultrasonic extraction at 55 ℃ for 30 min to ensure complete disruption of the coating carrier and complete dissolution of the target analyte; immediately after extraction, rapid cooling to room temperature in an ice-water bath terminated thermal degradation, standardized the system temperature, reduced solvent evaporation and systematic errors, and after volume adjustment, filtered through a 0.22 μm organic filter membrane. The filtrate was then ready for analysis.

[0023] 1.6 Chromatographic conditions A C18 column (4.6 mm × 150 mm, 5 μm) was used. The mobile phase was methanol:water = 80:20 (V / V), the flow rate was 1.0 mL / min, the column temperature was 26–28 ℃, the detection wavelength was 265 nm, and the injection volume was 20 μL. The mobile phase was degassed using a 0.45 μm filter. After detection, the column was maintained using a segmented gradient wash: first, 10% methanol for 1 h, then 100% methanol for 1 h. This effectively removed residual impurities, stabilized the baseline, and extended column life.

[0024] 1.7 Measurement and Calculation After the instrument baseline stabilizes, the sample is injected for detection. The mass fraction of 25-hydroxycholecalciferol in the sample is calculated using the external standard method, and the result is retained to three significant figures.

[0025] 2. Results and Analysis 2.1 Linear Relationship A series of standard working solutions with concentrations of 2.0, 4.0, 6.0, 8.0, and 10.0 μg / mL were prepared and injected for detection under optimized chromatographic conditions. A standard curve was fitted with solution concentration as the x-axis and chromatographic peak area as the y-axis, yielding the regression equation Y = 86415X + 986.2, with a coefficient of determination R0. 2 =0.9999. The results show that 25-hydroxycholecalciferol exhibits excellent linearity in the concentration range of 2.0–10.0 μg / mL, which can fully meet the requirements for accurate quantitative detection of trace coated samples.

[0026] 2.2 Solvent system optimization effect This invention employs the industry-standard 80:20 methanol-water system without altering the publicly disclosed ratio, focusing on optimizing the accompanying pretreatment process. Traditional methods use only the solvent but lack standardized temperature control, cooling, and light-protection procedures, resulting in incomplete extraction, peak tailing, low impurity separation, and large data fluctuations. This invention, through optimized integrated processes, improves extraction recovery by approximately 4.2% and impurity separation by approximately 15.6% compared to traditional extensive processes. It significantly enhances carrier dissolution, reduces target analyte degradation, improves chromatographic separation, and significantly outperforms conventional operations in detection stability.

[0027] 2.3 Optimization of Pretreatment Process Parameters To determine the optimal extraction conditions, single variables were fixed, and single-factor gradient optimization experiments were conducted on extraction temperature, ultrasonic duration, etc. Six parallel samples were set up in each group. The optimal process parameters suitable for the coated samples were screened by quantitative data.

[0028] 2.3.1 Extraction Temperature Optimization With a fixed ultrasound duration of 30 min and natural cooling, five temperature gradient tests were conducted at 35 ℃, 45 ℃, 55 ℃, 60 ℃, and 65 ℃. The test results under each temperature condition are shown in Table 1.

[0029] Table 1. Detection results of samples at different extraction temperatures:

[0030] As shown in Table 1, under low-temperature conditions of 35–45 °C, the coating carrier swells and lyses insufficiently, resulting in incomplete dissolution of the target analyte and a recovery rate of only 92.14%–95.37%. The RSD of parallel samples was greater than 1.40%, indicating significant data fluctuations. At 55 °C, the extraction recovery rate reached 98.96%, the RSD of parallel samples decreased to 0.76%, and the repeatability was good. Simultaneously, the degradation rate of the target analyte was only 0.71%, with a chromatographic resolution of 3.02 and a tailing factor of 1.02, indicating symmetrical peaks and excellent separation. At 60 °C, the recovery rate was slightly higher (99.12%), but the degradation rate increased slightly. Upon heating to 65 °C, the thermal degradation of the target analyte intensified dramatically, with the degradation rate soaring to 1.54%, and the chromatographic resolution decreased to 2.85%, indicating significant interference from impurities. Considering both extraction efficiency and substance stability, 55 °C was determined to be the optimal extraction temperature. Existing standards and publicly available literature do not provide a specific 55 °C isothermal cell disruption process for coated samples, making this a unique optimization parameter for this method.

[0031] 2.3.2 Optimization of Ultrasound Duration With the optimal extraction temperature fixed at 55 ℃ and natural cooling, ultrasonic duration gradient experiments were conducted at 10 min, 20 min, 30 min, 40 min, and 50 min. The experimental results are shown in Table 2.

[0032] Table 2. Sample detection results for different ultrasound durations:

[0033] Table 2 shows that when the ultrasonic extraction time is less than 30 min, the carrier cell disruption is incomplete, and the recovery rate increases significantly with time. Extraction reaches saturation at 30 min, with a stable recovery rate of 98.92%, exhibiting optimal repeatability and low degradation rate. Further extending the extraction time to 40-50 min does not significantly improve the recovery rate, but the target material remains under high temperature for an extended period, resulting in a maximum degradation rate of 1.32%. Therefore, 30 min is selected as the optimal ultrasonic extraction time.

[0034] 2.3.3 Effect of process on the stability of the chromatographic system The stability of the chromatographic system was compared by using a conventional uncontrolled temperature process and the optimized process of this invention for 20 consecutive injections. The results are shown in Table 3.

[0035] Table 3. Stability results of chromatographic systems for different processes:

[0036] As shown in Table 3, the optimized process of this invention can significantly reduce baseline noise and drift, with the peak area RSD decreasing from 1.42% to 0.45%, and the stability of the chromatographic system greatly improved, making it suitable for continuous detection of batch samples. In summary, the 55 ℃ isothermal ultrasonication for 30 min + ice-water shaping and cooling coupling process established in this study is an innovative process specifically for coated samples, effectively filling a technological gap in the industry.

[0037] 2.4 Spike Recovery Test Three spiking gradients (low, medium, and high) were set up, and the experimental results are shown in Table 4.

[0038] Table 4. Spike recovery results:

[0039] The results showed that the recoveries of each gradient spike were stable at 97.5%–98.2%, with an average recovery of 97.9%. The RSD of all parallel samples was less than 1.0%, indicating that the method has high accuracy, strong resistance to matrix interference, and is suitable for the detection of trace coated samples.

[0040] 2.5 Method Precision Test Six samples of the same batch of coated material were prepared in parallel for testing, and the precision results are shown in Table 5.

[0041] Table 5. Precision test results:

[0042] The experimental results show that the maximum absolute difference of the six parallel samples is only 0.0001%, which is far below the limit requirement of 1.0% for the dual standards. The overall precision of the method is RSD=0.42%, which is far better than the standard index of ≤10%, and the repeatability is excellent.

[0043] 2.6 Sample solution stability test The prepared sample solution was sealed and placed at room temperature away from light, and tested by the instrument at 0 h, 2 h, 4 h and 6 h respectively. The stability results are shown in Table 6.

[0044] Table 6. Stability test results:

[0045] When the sample solution is placed at room temperature in the dark for 6 hours, the detection content does not decrease significantly, and the RSD at each time point is less than 1.0%, indicating excellent solution stability. It can meet the needs of continuous batch testing in the laboratory and has a much higher fault tolerance than traditional testing processes.

Claims

1. A method for determining the content of 25-hydroxycholecalciferol in a feed additive, characterized in that, The steps include the following: S1. Prepare the standard working solution; S2. Preparation of the test sample solution: Weigh the sample and place it in a methanol aqueous solution, sonicate at 55℃ for 30 min, cool and then filter to obtain the test sample solution; S3. Liquid chromatography detection: The standard working solution and the sample solution to be tested are injected separately for detection. The retention time is used for qualitative analysis, and the peak area external standard method is used for quantitative analysis to obtain the content of 25-hydroxycholecalciferol in the sample solution to be tested.

2. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 1, characterized in that, The standard working solution is prepared by dissolving a standard of 25-hydroxycholecalciferol aqueous compound in an aqueous methanol solution.

3. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 1, characterized in that, In step S2, the cooling method is ice-water bath cooling.

4. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 1, characterized in that, Steps S1 and S2 must be performed in the dark.

5. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 1, characterized in that, In step S2, the volume ratio of methanol to water in the methanol-water solution is 80:

20.

6. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 1, characterized in that, In step S2, a 0.22 μm organic filter membrane is used for filtration.

7. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 1, characterized in that, The liquid chromatography conditions for step S3 are as follows: C18 column, column size 4.6 mm × 150 mm, 5 μm; mobile phase is methanol-water solution; flow rate 1.0 mL / min, column temperature 26~28 ℃, detection wavelength 265 nm, injection volume 20 μL.

8. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 7, characterized in that, The volume ratio of methanol to water in the mobile phase is 80:

20.

9. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 7, characterized in that, The mobile phase was pretreated by degassing through a 0.45 μm filter membrane.

10. The method for determining the content of 25-hydroxycholecalciferol in a feed additive according to claim 1, characterized in that, After step S3 is completed, the column is maintained by segmented gradient rinsing: first rinse with 10% methanol aqueous solution for 1 hour, then rinse with pure methanol for 1 hour.

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