A method for determining the ortho value iron content in EDDHA-Fe chelate by HPLC

Abstract

This invention discloses an HPLC method for determining the ortho-O iron content in EDDHA-Fe chelates, comprising the following steps: S1. Sample pretreatment: S11. The sample is first extracted with an aqueous solution, followed by solid-liquid separation; S12. The liquid phase is purified by passing it through a strong acid cation exchange resin to obtain a purified solution; S13. EDTA salt is added to obtain a test solution; S2. HPLC detection: A C18 reversed-phase column is used, with gradient elution using mobile phase A and mobile phase B; mobile phase A is a 0.05-0.15% (w / w) aqueous solution of phosphoric acid with a pH of 2.5-3.0; mobile phase B is methanol. This invention, through optimized chromatographic conditions and innovative sample pretreatment, establishes a high-resolution, interference-resistant method for O-O detection, solving the problems of insufficient resolution, interference ion influence, and inaccurate quantification in existing methods.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry detection technology, specifically relating to a method for determining the ortho-position iron content in EDDHA-Fe chelates by HPLC. Background Technology

[0002] EDDHA-Fe (ethylenediamine di-o-hydroxyphenylacetic acid iron) chelate is a highly efficient iron chelating compound (including but not limited to EDDHA-Fe, EDDHA-FeNa, EDDHA-FeK, etc.) and is widely used to correct iron deficiency in plants. However, its chemical synthesis inevitably produces various spatial isomers, among which the main active ingredient is the ortho-ortho isomer (oo), while impurities such as the ortho-para isomers (o, p) have very low activity, affecting product efficacy. The structures of the ortho-ortho isomer (oo) and the ortho-para isomer (op) are shown in Formula 1.

[0003] Formula 1 Accurate determination of oo content is crucial for product quality control. Currently, the industry's testing of the active ingredient in EDDHA-Fe chelate products generally suffers from the following deficiencies: 1. Insufficient chromatographic separation efficiency; the resolution between oo and op is less than 1.5, leading to quantitative errors. 2. Ca was not effectively removed. 2+ Mg 2+ Competitive interfering ions can affect the stability of chelated iron. 3. Lack of targeted antioxidant and light-protection measures can easily lead to photolysis of EDDHA-Fe chelates or the release of free Fe. 3+ This process reduces detection accuracy. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the ortho-position iron content in EDDHA-Fe chelates by HPLC, in order to overcome the shortcomings of the prior art.

[0005] The objective of this invention is achieved through the following technical solution: A method for determining the ortho-position iron content in EDDHA-Fe chelates by HPLC includes the following steps: S1. Sample pretreatment: S11. The sample is first extracted with an aqueous solution, and then solid-liquid separation is performed; S12. The liquid phase after solid-liquid separation is purified by passing it through a strong acid cation exchange resin to obtain a purified liquid; S13. Add EDTA salt as a metal ion masking agent to the purified solution to obtain the test solution; S2. HPLC detection: The content of ortho-position iron in the EDDHA-Fe chelate in the test solution was detected by HPLC; the chromatographic conditions were as follows: A C18 reversed-phase column was used for gradient elution with mobile phases A and B. The mobile phase A is a phosphoric acid aqueous solution with a mass percentage of 0.05-0.15% and a pH of 2.5-3.0; the mobile phase B is methanol. The gradient elution procedure is as follows: In the first stage, the volume fraction of mobile phase B gradually increases from 15-25% to 55-65%, with the remainder being mobile phase A; the duration of the first stage is 8-12 minutes. In the second stage, the volume fraction of mobile phase B is 55-65%, and the remainder is mobile phase A; the duration of the second stage is 8-12 minutes. In the third stage, the volume fraction of mobile phase B is 15-25%, and the remainder is mobile phase A; the duration of the third stage is 8-12 minutes. The external standard method was used to quantitatively analyze the ortho-position iron content in the EDDHA-Fe chelate.

[0006] Preferably, the aqueous solution in step S1 is any one selected from pure water and phosphoric acid aqueous solution with a pH of 2.5 to 3.0.

[0007] Preferably, the amount of the aqueous solution used in step S1 is 40 to 1000 times the mass of the sample.

[0008] Preferably, the flow rate of the mobile phase in step S2 is 0.8~1.2 mL / min, and the column temperature is 28~32℃; the detection wavelength is the maximum absorption wavelength of the EDDHA-Fe chelate.

[0009] Preferably, the strong acid type cation exchange resin is selected from either sulfonic acid type or phosphoric acid type.

[0010] Preferably, the sulfonic acid type cation exchange resin is Dowex 50WX8, and the flow rate is 0.8~1.2 mL / min.

[0011] Preferably, the extraction in step S1 is performed by ultrasonic extraction, with an ultrasonic power of 100-200W and a time of 5-15min.

[0012] Preferably, the amount of EDTA salt used is such that the concentration of EDTA salt in the test solution is 1~10 μg / mL.

[0013] Preferably, the C18 chromatographic column has an inner diameter of 4.6 mm and a length of 250 mm, with a packing particle size of 5 μm; The detection wavelength in step S2 is 280nm.

[0014] Preferably, the chromatographic conditions in step S2 are as follows: A C18 column was used, with gradient elution of mobile phases A and B. The mobile phase flow rate was 1 mL / min, the column temperature was 30 °C, the detection wavelength was 280 nm, and the injection volume was 20 μL. The mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid with a pH of 2.5–3.0; the mobile phase B is methanol. The gradient elution procedure is as follows: During the first 10 minutes, the volume fraction of mobile phase B increased from 20% to 60%, with the remainder being mobile phase A. During the 10-20 min period, the volume fraction of mobile phase B was 60%, with the remainder being mobile phase A. Between 20 and 21 minutes, the volume fraction of mobile phase B decreased from 60% to 20%, with the remainder being mobile phase A. Between 21 and 30 minutes, the volume fraction of mobile phase B was 20%, with the remainder being mobile phase A. The external standard method was used to quantitatively analyze the ortho-position iron content in the EDDHA-Fe chelate.

[0015] This invention establishes a high-resolution, interference-resistant method for detecting oo by optimizing chromatographic conditions and innovating sample pretreatment, solving the problems of insufficient resolution, interference ion influence, and inaccurate quantification in existing methods. Attached Figure Description

[0016] Figure 1 This is the chromatogram of the sample from Example 1 obtained by HPLC detection. Detailed Implementation

[0017] This application provides a method for determining the ortho-position iron content in EDDHA-Fe chelates by HPLC, comprising the following steps: S1. Sample pretreatment: S11. The sample is first extracted with an aqueous solution, followed by solid-liquid separation. EDDHA-Fe chelates have good water solubility; therefore, this application uses an aqueous solution as the extraction solvent to ensure the EDDHA-Fe chelates in the sample are fully dissolved in water. To avoid insoluble impurities interfering with subsequent operations (specifically, clogging of the subsequent ion exchange resin), the extracted material is subjected to solid-liquid separation to remove insoluble impurities. The preferred solid-liquid separation method is filtration using a 0.22 μm microfiltration membrane.

[0018] Ultrasonic-assisted extraction is preferred during extraction, and more preferably, the ultrasonic power is 100~200w and the time is 5~15min.

[0019] Preferably, to prevent sample degradation, extraction is performed under light-protected conditions.

[0020] Preferably, the aqueous solution can be pure water, a phosphoric acid aqueous solution with a pH of 2.5 to 3.0, etc. The amount of aqueous solution used is 40 to 1000 times the sample mass.

[0021] Preferably, extraction is performed using an aqueous phosphoric acid solution, which stabilizes the pH of the extraction system and the chelate, and effectively inhibits Fe. 3+ Hydrolysis improves extraction efficiency.

[0022] S12. The liquid phase after solid-liquid separation is purified by passing it through a strong acid cation exchange resin to obtain a purified liquid; Strong acid cation exchange resins for Ca 2+ Mg 2+ Interfering ions have a good removal effect and can reduce Ca. 2+ Mg 2+ To reduce interference from impurities and improve measurement accuracy.

[0023] Preferably, the strong acid type cation exchange resin is selected from any one of sulfonic acid type, phosphoric acid type, etc. More preferably, it is a sulfonic acid type cation exchange resin, for the following reasons: 1. Common impurity ions in EDDHA-Fe chelate samples (such as Ca) 2+ Mg 2+ K + Na + All of them carry a positive charge at ambient pH. Sulfonic acid resins have strong acidic sulfonic acid groups (-SO3H), which can completely dissociate over a wide pH range (especially suitable for acidic sample solutions). They exhibit high capacity and high selectivity in exchanging these interfering cations, thereby efficiently removing them and avoiding their competition for coordination with EDDHA or their impact on the chromatographic baseline.

[0024] 2. Does not damage the target analyte: the chelate structure of the EDDHA-Fe chelate (Fe 3+ (Tightly encapsulated by organic ligands) it is electrically neutral or negatively charged and will not be retained by cation exchange resins. Therefore, the purification process can selectively remove impurities while allowing the target analyte, EDDHA-Fe chelate, to pass through quickly with almost no adsorption, resulting in high recovery rates and avoiding losses or peak tailing caused by resin adsorption.

[0025] More preferably, the sulfonic acid type cation exchange resin is Dowex 50WX8, and the flow rate is 0.8~1.2 mL / min.

[0026] S13. Add EDTA salt as a metal ion masking agent to the purification solution to obtain the test solution; the EDTA salt can be disodium EDTA and / or dipotassium EDTA. EDTA salt can effectively mask metal ions and prevent metal ions from interfering with the measurement, thereby further improving the accuracy of the test.

[0027] Preferably, the amount of EDTA salt used is such that the concentration of EDTA salt in the test solution is 1~10 μg / mL.

[0028] When the sample is a raw material, the EDDHA-Fe chelate has high purity. The above pretreatment method can remove impurities as much as possible and reduce interference. The HPLC method can effectively separate OO and OP in the EDDHA-Fe chelate.

[0029] After extraction and ion exchange resin treatment, the sample volume generally changes. Therefore, after extraction and the addition of disodium EDTA, the volume can be adjusted to a precise, known volume using the extraction reagent for convenient content calculation. To improve detection efficiency, a certain volume of the liquid phase can be used for ion exchange resin purification in step S12; it is not necessary to use the entire liquid phase for ion exchange resin purification.

[0030] To minimize the impact of light on EDDHA-Fe chelates, it is preferable to store the sample solution in the dark before HPLC detection.

[0031] S2. HPLC detection: The test solution was analyzed using HPLC; the chromatographic conditions were as follows: A C18 reversed-phase column was used, with gradient elution using mobile phases A and B. The detection wavelength was the maximum absorption wavelength of the EDDHA-Fe chelate, which was 280 nm. At this wavelength, the response value of oo was more than 30% higher than that of op.

[0032] Mobile phase A is a phosphoric acid aqueous solution with a mass percentage of 0.05–0.15% and a pH of 2.5–3.0; using a phosphoric acid aqueous solution with a pH of 2.5–3.0 can effectively inhibit Fe3+ Hydrolysis enhances the symmetry of chromatographic peaks (tailing factor T = 0.95-1.05).

[0033] Mobile phase B is methanol, preferably chromatographic grade; the flow rate of the mobile phase is 0.8~1.2 mL / min, and the column temperature is 28~32℃; The gradient elution procedure is as follows: In the first stage, the volume fraction of mobile phase B gradually increases from 15-25% to 55-65%, with the remainder being mobile phase A; the duration of the first stage is 8-12 minutes. In the second stage, the volume fraction of mobile phase B is 55-65%, and the remainder is mobile phase A; the duration of the second stage is 8-12 minutes. In the third stage, the volume fraction of mobile phase B is 15-25%, and the remainder is mobile phase A; the duration of the third stage is 8-12 minutes.

[0034] In the first stage, the methanol ratio is linearly increased, enabling rapid separation of oo and op. The second stage ensures that all stronger impurity components in the sample are thoroughly eluted from the column, while simultaneously cleaning the column to prepare it for rapid equilibration in the next stage. The third stage restores the initial ratio and equilibrates the column.

[0035] The C18 reversed-phase column has a uniform stationary phase bonding density and specific separation capability for oo and op. Validated by the SN-CEN / TS 15452:2006 standard, the resolution Rs≥1.5, meeting the baseline separation requirements for isomers.

[0036] Preferably, the C18 column has an inner diameter of 4.6 mm and a length of 250 mm, with a packing particle size of 5 μm.

[0037] This application employs the external standard method to quantitatively analyze the ortho-position iron content in EDDHA-Fe chelates. The external standard method is simpler to operate than the internal standard method.

[0038] As those skilled in the art will understand, the external standard method includes the step of preparing a standard curve, as follows: Prepare O0 standard solutions (specifically O,O-EDDHA-FeNa standard) with concentrations of 5, 10, 20, 50, and 100 μg / mL, using the same solvent as the sample extraction reagent. Inject the samples under the chromatographic conditions described above, and plot a standard curve using peak area (A) against concentration (C, μg / mL). One preferred embodiment yields a regression equation of A = 5000C + 100, with a correlation coefficient R0. 2 ≥0.999.

[0039] In a preferred embodiment, the chromatographic conditions for step S2 are as follows: A C18 column was used, with gradient elution of mobile phases A and B. The mobile phase flow rate was 1 mL / min, the column temperature was 30 °C, the detection wavelength was 280 nm, and the injection volume was 20 μL. Mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid with a pH of 2.5–3.0; mobile phase B is methanol. The gradient elution procedure is as follows: During the first 10 minutes, the volume fraction of mobile phase B increased from 20% to 60%, with the remainder being mobile phase A. During the 10-20 min period, the volume fraction of mobile phase B was 60%, with the remainder being mobile phase A. Between 20 and 21 minutes, the volume fraction of mobile phase B decreased from 60% to 20%, with the remainder being mobile phase A. Between 21 and 30 minutes, the volume fraction of mobile phase B was 20%, with the remainder being mobile phase A. The external standard method was used to quantitatively analyze the ortho-position iron content in EDDHA-Fe chelates.

[0040] Method validation system

[0041] 1. Linearity and Sensitivity (1) Linear range: 5~100μg / mL, with good linearity (R0). 2 ≥0.999).

[0042] (2) Limit of detection (LOD): 0.5 μg / mL (S / N=3), limit of quantitation (LOQ): 1.0 μg / mL (S / N=10).

[0043] 2. Precision and accuracy (1) Repeatability: The same reference solution (specifically, o,o-EDDHA-FeNa standard solution with a concentration of 20 μg / mL) was injected 6 times consecutively, and the retention time RSD of o was 0.8% and the peak area RSD was 1.2%, which showed excellent precision.

[0044] (2) Spike recovery rate: Accurately weigh several portions of EDDHA-FeNa sample with known oo content (specifically, the sample of Example 1, used as blank matrix), and accurately add oo-EDDHA-FeNa standard at three concentration levels of 80%, 100%, and 120%, respectively. Process and determine according to the sample pretreatment method (S1) and chromatographic conditions (S2) of Example 1 of this invention. Calculate the spike recovery rate based on the difference between the measured total amount and the sample background amount. The spike recovery rate is 98~102%, and RSD≤1.5%.

[0045] 3. Stability and Durability (1) Solution stability: The sample solution (specifically the sample solution of Example 1) was placed in the dark for 24 hours, and the peak area RSD was 1.0%, indicating good stability.

[0046] (2) Column durability: After 200 injections on the same column, the resolution Rs=1.6 (initial Rs=1.8) and the column efficiency decreases by <10%, which meets the long-term detection requirements.

[0047] In summary, compared with the prior art, this application has the following beneficial effects: 1. Multi-stage anti-interference pretreatment technology: Strong acid cation exchange resin is used to effectively remove metal ion interference, and then EDTA salt is used to stabilize chelates and mask metal ion interference, which significantly improves detection stability.

[0048] 2. Precise separation technology for isomers: By selecting C18 columns and optimizing mobile phase and gradient elution, baseline separation of oo and op is achieved (Rs≥1.5), solving the core problem of insufficient separation in existing methods.

[0049] Through the aforementioned innovations, this patent constructs a complete detection system for oo in EDDHA-Fe chelates. The technical solution is scientifically rigorous, the verification data is detailed, and it complies with the Chinese Pharmacopoeia and international testing standards, possessing significant industrial application value.

[0050] Example 1

[0051] 1. Standard Curve Creation Weigh 50 mg of o,o-EDDHA-FeNa standard (accurate to 0.0001 g), and dilute to 50 mL with 0.1% phosphoric acid aqueous solution (pH 2.5) to prepare a 1000 μg / mL stock solution. Then, serially dilute to a series of concentrations of 5~100 μg / mL (specifically 5, 10, 20, 50, and 100 μg / mL).

[0052] The standard solution was analyzed by HPLC under the following chromatographic conditions.

[0053] A C18 column was used, with gradient elution of mobile phases A and B. The mobile phase flow rate was 1 mL / min, the column temperature was 30 °C, the detection wavelength was 280 nm, and the injection volume was 20 μL. Mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid with a pH of 2.5–3.0; mobile phase B is methanol. The gradient elution procedure is as follows: During the first 10 minutes, the volume fraction of mobile phase B increased from 20% to 60%, with the remainder being mobile phase A. During the 10-20 min period, the volume fraction of mobile phase B was 60%, with the remainder being mobile phase A. Between 20 and 21 minutes, the volume fraction of mobile phase B decreased from 60% to 20%, with the remainder being mobile phase A. Between 21 and 30 minutes, the volume fraction of mobile phase B was 20%, with the remainder being mobile phase A.

[0054] A standard curve was plotted using peak area (A) against concentration (C, μg / mL), yielding the regression equation A = 5000C + 100, and the correlation coefficient R0. 2 ≥0.999.

[0055] 2. Sample testing Weigh 0.1g of commercially available EDDHA-FeNa product (accurate to 0.0001g), place it in a 50mL brown volumetric flask, add 40mL of 0.1% phosphoric acid aqueous solution (pH 2.5) to dissolve, sonicate for 10min (power 150W), bring the volume to 50mL, filter through a 0.22μm nylon filter membrane to remove insoluble impurities; Take 20 mL of filtrate and purify it by passing it through a sulfonic acid type cation exchange resin (Dowex 50WX8, column length 10 cm) at a flow rate of 1 mL / min to obtain the purified solution; Add 50 μL of 0.05% EDTA disodium solution, then dilute to 25 mL with 0.1% phosphoric acid aqueous solution (pH 2.5) to obtain the test solution; let stand in the dark for 10 min.

[0056] Inject 20 μL of the test solution under the chromatographic conditions described above, and record the chromatogram at a wavelength of 280 nm. Figure 1 As shown, the retention time of oo is approximately 8.5 min, the retention time of op is approximately 10.2 min, and the resolution Rs = 1.7.

[0057] The concentration corresponding to the peak area of ​​the sample was calculated based on the standard curve, and the iron oo content in the ortho position was calculated. The oo content in this sample was 86.5% (n=3, RSD=1.0%).

[0058] Example 2 1. Sample testing Compared to Example 1, the sample was replaced with an EDDHA-FeNa formulation, and the sample processing method is as follows: Weigh 10 tablets, grind them, weigh 0.2g, add 40mL of 0.1% phosphoric acid aqueous solution (pH 2.5), extract by ultrasonication (150W power) for 20min, make up to 50mL, filter through a 0.22μm nylon filter membrane to remove insoluble impurities; Take 20 mL of filtrate and purify it by passing it through a sulfonic acid type cation exchange resin (Dowex 50WX8, column length 10 cm) at a flow rate of 1 mL / min to obtain the purified solution; Add 50 μL of 0.05% EDTA disodium solution, then dilute to 25 mL with 0.1% phosphoric acid aqueous solution (pH 2.5) to obtain the test solution; let stand in the dark for 10 min.

[0059] The remaining steps are the same as in Example 1.

[0060] The test results were as follows: Three parallel determinations were performed, and the average content of the ortho-ortho isomer (oo) in the EDDHA-FeNa tablets was calculated to be 6.5% (w / w), with a relative standard deviation (RSD) of 1.5%. The retention time of the oo chromatographic peak was stable (RSD = 0.3%), and the resolution (Rs) with the ortho-para isomer (op) was 1.6.

[0061] 2. Interference Verification Adding 100 ppm Ca²⁺ to the sample and treating it according to the above steps resulted in a change of <0.5% in the peak area of ​​oo, indicating a significant removal effect of interfering ions.

[0062] Comparative Example 1 (without ion exchange resin purification) 1. Sample preparation: Except for omitting step S12 (ion exchange resin purification), the remaining steps are exactly the same as in Example 1. That is, the filtrate after filtration through a 0.22 μm filter membrane in S11 is directly used for subsequent addition of disodium EDTA and volume adjustment.

[0063] 2. Test Results: Resolution: Rs = 1.5 (similar to 1.7 in Example 1, indicating that the separation is acceptable).

[0064] Spiked recovery: When the same concentration of oo standard was added to a known sample, the average recovery rate was only 92.5% (significantly lower than the recovery rate range of 98~102% in the method validation of this invention), and the RSD increased to 3.5%.

[0065] Conclusion: Although chromatographic separation was still acceptable after omitting the ion exchange step, the recovery rate of the target analyte was significantly reduced and the precision deteriorated because interfering metal ions in the sample matrix were not removed. This proves that the purification step is crucial for ensuring quantitative accuracy.

[0066] Comparative Example 2 (using an isocratic elution procedure) The sample processing steps are the same as in the example.

[0067] 1. Chromatographic conditions: Except for changing the gradient elution program to isocratic elution (mobile phase B with a constant methanol volume fraction of 40% and mobile phase A with 60%), all other chromatographic conditions (column, flow rate, column temperature, detection wavelength, etc.) were the same as in Example 1. The total run time was 30 minutes.

[0068] 2. Test Results: Resolution: The chromatographic peaks of oo and op overlapped severely, and the resolution Rs was only 0.8, making baseline separation impossible.

[0069] Quantitative results: Due to peak overlap, the peak area of ​​oo cannot be accurately integrated, resulting in extremely poor repeatability of the quantitative results (RSD > 10%) and a huge deviation from the known content.

[0070] Conclusion: Conventional isocratic elution cannot solve the separation problem of oo and op, which fully demonstrates the necessity of the specific gradient elution procedure of this invention for achieving accurate separation of isomers.

[0071] Comparative Example 3 (without added EDTA disodium stabilizer) Sample processing: Except for omitting step S13 (not adding EDTA disodium solution), the other sample pretreatment steps are exactly the same as in Example 1.

[0072] 2. Test Results: Solution stability: The prepared test solution was placed under light-protected conditions for 0 hours, 2 hours, and 4 hours before being injected for analysis. The peak area of ​​oo decreased by 8.5% within 4 hours (the RSD of the solution in Example 1 was only 1.0% within 24 hours).

[0073] Content determination: The results were similar to those in Example 1 when using freshly prepared solutions. However, when using solutions that had been left to stand for 2 hours, the content was found to be about 5% lower.

[0074] Conclusion: Without the addition of disodium EDTA, the stability of the EDDHA-Fe chelate in the test solution decreased significantly, and it was prone to degradation or transformation over time, leading to inaccurate test results. This demonstrates the necessity of adding EDTA as a stabilizer and the excellent stability of this method.

[0075] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if these modifications and modifications of the invention fall within the scope of the claims and their equivalents, the invention is also intended to include these modifications and modifications.

Claims

1. A method for determining the ortho value iron content in EDDHA-Fe chelate by HPLC method, characterized in that, Includes the following steps: S1. Sample pretreatment: S11. The sample is first extracted with an aqueous solution, and then solid-liquid separation is performed; S12. The liquid phase after solid-liquid separation is purified by passing it through a strong acid cation exchange resin to obtain a purified liquid; S13. Add EDTA salt as a metal ion masking agent to the purified solution to obtain the test solution; S2. HPLC detection: The content of ortho-position iron in the EDDHA-Fe chelate in the test solution was detected by HPLC; the chromatographic conditions were as follows: A C18 reversed-phase column was used for gradient elution with mobile phases A and B. The mobile phase A is a phosphoric acid aqueous solution with a mass percentage of 0.05-0.15% and a pH of 2.5-3.0; the mobile phase B is methanol. The gradient elution procedure is as follows: In the first stage, the volume fraction of mobile phase B gradually increases from 15-25% to 55-65%, with the remainder being mobile phase A; the duration of the first stage is 8-12 minutes. In the second stage, the volume fraction of mobile phase B is 55-65%, and the remainder is mobile phase A; the duration of the second stage is 8-12 minutes. In the third stage, the volume fraction of mobile phase B is 15-25%, and the remainder is mobile phase A; the duration of the third stage is 8-12 minutes. The external standard method was used to quantitatively analyze the ortho-position iron content in the EDDHA-Fe chelate.

2. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim , characterized in that, The aqueous solution in step S1 is any one selected from pure water and phosphoric acid aqueous solution with a pH of 2.5 to 3.

0.

3. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 3, characterized in that, The amount of the aqueous solution used in step S1 is 40 to 1000 times the mass of the sample.

4. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 1, characterized in that, In step S2, the flow rate of the mobile phase is 0.8~1.2 mL / min, and the column temperature is 28~32℃; the detection wavelength is the maximum absorption wavelength of the EDDHA-Fe chelate.

5. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 1, characterized in that, The strong acid type cation exchange resin is selected from either sulfonic acid type or phosphoric acid type.

6. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 5, characterized in that, The sulfonic acid type cation exchange resin is Dowex 50WX8, and the flow rate is 0.8~1.2 mL / min.

7. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 1, characterized in that, The extraction in step S1 is performed using ultrasonic extraction, with an ultrasonic power of 100-200W and a time of 5-15 minutes.

8. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 1, characterized in that, The amount of EDTA salt used is such that the concentration of EDTA salt in the test solution is 1~10 μg / mL.

9. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 1, characterized in that, The C18 chromatographic column has an inner diameter of 4.6 mm and a length of 250 mm, with a packing particle size of 5 μm. The detection wavelength in step S2 is 280nm.

10. The method for determining the ortho-position iron content in EDDHA-Fe chelate by HPLC as described in claim 1, characterized in that, The chromatographic conditions described in step S2 are as follows: A C18 column was used, with gradient elution of mobile phases A and B. The mobile phase flow rate was 1 mL / min, the column temperature was 30 °C, the detection wavelength was 280 nm, and the injection volume was 20 μL. The mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid with a pH of 2.5–3.0; the mobile phase B is methanol. The gradient elution procedure is as follows: During the first 10 minutes, the volume fraction of mobile phase B increased from 20% to 60%, with the remainder being mobile phase A. During the 10-20 min period, the volume fraction of mobile phase B was 60%, with the remainder being mobile phase A. Between 20 and 21 minutes, the volume fraction of mobile phase B decreased from 60% to 20%, with the remainder being mobile phase A. Between 21 and 30 minutes, the volume fraction of mobile phase B was 20%, with the remainder being mobile phase A. The external standard method was used to quantitatively analyze the ortho-position iron content in the EDDHA-Fe chelate.

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