Quantitative analysis method for oxidation state proportion of Fe < 2 + > and Fe < 3 + > in nano ferrite particles and application

Abstract

The invention belongs to the technical field of content analysis of ferrous ions and iron ions in nano ferrite, and particularly relates to a quantitative analysis method for the oxidation state proportion of Fe < 2 + > and Fe < 3 + > in nano ferrite particles and application. According to the quantitative analysis method for the oxidation state proportion of Fe < 2 + > and Fe < 3 + > in the nano ferrite particles, the oxidation state proportion of Fe < 2 + > and Fe < 3 + > can be accurately measured under the condition of a conventional UV-Vis instrument through a set of wall breaking, protection, complete digestion and spectrum correction combined system designed according to the characteristics of nano ferrite, and the analysis precision close to that of Mssbauer spectroscopy is achieved; according to the method, the accuracy and repeatability of valence analysis of the nano ferrite are remarkably improved, the requirements of instruments and environmental conditions on testing are effectively reduced, and the method can be suitable for multiple dosage forms such as a water phase, an oil phase, freeze-dried powder and PEG or SiO2 coating, and is wide in application range, high in technical accessibility and suitable for industrial production. And a feasible technical means is provided for the quality control of the nano ferrite medicine.

Description

Technical Field

[0001] This invention belongs to the technical field of ferrous and ferric ion content analysis in nano-ferrites, specifically relating to the analysis of Fe content in nano-ferrite particles. 2+ and Fe 3+ A quantitative analysis method for the proportion of oxidation states. Background Technology

[0002] Nanoferrites (Fe3O4, γ-Fe2O3, etc.) are widely used in magnetic resonance imaging agents, magnetic nanomedicines, magnetic navigation and catalysts. One of their key quality attributes (CQA) is Fe. 2+ / Fe 3+ The oxidation state ratio directly affects the following indicators: 1. Magnetic resonance T1 / T2 relaxation rate; 2. Magnetic saturation intensity; 3. Bulk phase and surface defect structure; 4. Clinical safety and stability. Therefore, the determination of Fe in nanoferrites... 2+ / Fe 3+ Determining the oxidation state ratio is a necessary task in relevant application fields.

[0003] Current methods for determining iron have the following main problems: 1. Traditional methods can only measure "total iron" (cannot separate Fe). 2+ / Fe 3 + Methods such as colorimetry and titration are only applicable to soluble iron ions and cannot measure the valence state of nanoparticles. 2. Titration methods that can measure valence states are not suitable for nanoparticles; traditional Fe... 2+ / Fe 3+ Titration is based on the premise of "complete dissolution + no oxidation", but nano-ferrite particles have: shell coating (oleic acid, PEG, SiO2, etc.), lattice Fe 2+ The characteristics of Fe, such as difficulty in color development and oxidation of 8-15% within 5 minutes in air, lead to serious deviations in titration results. 3. XPS (X-ray photoelectron spectroscopy) can only measure the surface 2-3 nm, failing to reflect the true Fe content of the entire 3-20 nm particle. 2+ / Fe 3+ The ratio can have an error exceeding 20%. 4. While Mössbauer spectroscopy can measure valence states, it cannot be used for QC or applied to pharmaceutical GMP quality control. Furthermore, it requires equipment and a liquid nitrogen system costing 1.5 million yuan, making it expensive. 5. The current pharmacopoeia does not include nano-ferrite Fe. 2+ / Fe 3+ The testing requirements necessitate outsourcing testing to companies (7–10 days), which fails to meet batch release efficiency standards. Summary of the Invention

[0004] This invention aims to provide Fe in nano-ferrite particles 2+ and Fe 3+A quantitative analysis method for oxidation state ratio is proposed to address the limitations of existing technologies in rapidly, accurately, and cost-effectively determining the true Fe content of nano-ferrites under ordinary UV-Vis (ultraviolet-visible) light and QC environments. 2+ / Fe 3+ Technical issues related to proportions.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: Provides Fe in nano-ferrite particles 2+ and Fe 3+ A quantitative analysis method for the oxidation state ratio, the method comprising: The nano-ferrite particle sample was dispersed in a cell-wall breaking solution for cell-wall breaking treatment. Under inert gas protection conditions, a complexing agent and Fe are added. 2+ Formation of complexes, enabling Fe 2+ It is thus protected from oxidation to Fe during the subsequent acid digestion process. 3+ ; The reaction system was initially digested under relatively mild acidic conditions; subsequently, any remaining particles that were not completely digested were thoroughly digested. The digested reaction system was used as a colorimetric system, and the absorbance of the system at two wavelengths, 510 nm and 630 nm, was measured simultaneously. The 510 nm wavelength represents the absorbance of Fe. 2+ The wavelength corresponding to the complexation peak, 630 nm, is the wavelength corresponding to the colloidal scattering sensitive region. The scattering intensity at 630 nm is corrected by subtracting the corrected value from the absorbance at 510 nm, yielding the Fe... 2+ True absorbance, through Fe 2+ True absorbance calculation Fe 2+ The concentration; In Fe 2+ After the concentration was accurately determined, hydroxylamine hydrochloride was added to the same colorimetric system to remove Fe from the sample. 3+ Completely reduced to Fe 2+ The absorbance measured again at 510 nm corresponds to Fe 2+ and+Fe 3+ The absorbance of total iron is used to calculate the concentration of total iron. The concentration of total iron is related to Fe. 2+ The concentration difference is Fe 3+ Concentration; ultimately, based on Fe 2+ with Fe 3+ The molar concentration of Fe in nano-ferrite can be used to calculate the Fe content. 2+ / Fe 3+ Oxidation state ratio.

[0006] Preferably, the process of dispersing the nano-ferrite particle sample in a cell-wall breaking solution for cell-wall breaking includes: dispersing the nano-ferrite sample in a cell-wall breaking solution composed of short-chain thiols and low-concentration hydrofluoric acid to form a preliminary reaction system, wherein the concentration of the short-chain thiols is controlled at 0.08–0.12 mol / L, the concentration of the hydrofluoric acid is controlled at 0.01–0.03 mol / L, and the pH of the reaction system is adjusted to approximately 3.5; after the sample in the reaction system is subjected to ultrasonic treatment at room temperature for 2–4 minutes, the outer oleic acid layer, PEG layer, or SiO2 shell will be effectively destroyed, thereby opening the channel for the colorimetric reagent to enter the particle lattice region.

[0007] Preferably, in the cell wall disruption process, the ratio of "sample mass: cell wall disruption solution volume" between the nano-ferrite particle sample and the cell wall disruption solution is "1:(100–500) (g:mL)".

[0008] Preferably, in the cell wall disruption process, the ratio of "sample mass: cell wall disruption solution volume" between the nano-ferrite particle sample and the cell wall disruption solution is "1:(150–300) (g:mL)".

[0009] Preferably, the complexing agent is 2,2'-bipyridine in solid powder form, with a particle size of 50–100 μm, used to cause Fe... 2+ The complexation is completed rapidly, forming Fe(bipy)3. 2+ Complex.

[0010] Preferably, the preliminary digestion of the reaction system under relatively mild acidic conditions includes: adding 0.1 mol / L hydrochloric acid to the reaction system and maintaining a temperature of about 37°C for several minutes to dissolve the nano-ferrite particles with smaller particle size and more surface defects in advance. The complete elimination of residual particles that have not been completely eliminated includes: adding an acidic mixture of 1.5 mol / L hydrochloric acid and 0.1 mol / L thioacetic acid to the reaction system, and heating to approximately 160°C under closed microwave conditions to completely destroy the nanoparticle lattice and eliminate the Fe... 2+ with Fe 3+ All of it enters the solution.

[0011] Preferably, when adding 0.1 mol / L hydrochloric acid to the reaction system, 2-5 mL of hydrochloric acid is added for every 50 mg of nano-ferrite particle sample; In the acidic mixed system consisting of 1.5 mol / L hydrochloric acid and 0.1 mol / L thioacetic acid added to the reaction system, each 50 mg of nano ferrite particle sample corresponds to 8–12 mL of acidic mixed system, and the molar ratio of hydrochloric acid to thioacetic acid in the acidic mixed system is approximately 15:1. The concentration of hydroxylamine hydrochloride solution added to the same colorimetric system is 0.5–2.0 mol / L; the volume percentage of the added hydroxylamine hydrochloride is 0.5–2.0% (v / v) of the total volume of the colorimetric system; Fe 3+ The molar ratio with hydroxylamine hydrochloride is not less than 1:10.

[0012] Preferably, the scattering intensity at a wavelength of 630 nm is corrected with a coefficient of 0.42.

[0013] This invention also provides Fe in any of the aforementioned nanoferrite particles. 2+ and Fe 3+ A quantitative analysis method for the oxidation state ratio in determining Fe in nano-ferrite samples 2+ and Fe 3+ Application in oxidation state ratios.

[0014] Preferably, the nano-ferrite sample is an aqueous phase, an oil phase, a lyophilized powder, or various organic / inorganic coated nano-ferrite samples.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. Fe in the nano-ferrite particles 2+ and Fe 3+ The quantitative analysis method for oxidation state ratio utilizes a combined system designed specifically for the characteristics of nano-ferrites, including cell disruption, protection, complete digestion, and spectral correction, to enable Fe... 2+ / Fe 3+ 1. The oxidation state ratio can be accurately determined under conventional UV-Vis instrument conditions, achieving analytical precision close to that of Mössbauer spectroscopy. 2. The two-step digestion technique employed in this method considers both the initial dissolution characteristics of nanoparticles under low acidity conditions and the complete digestion of various coated nano-ferrites through a closed microwave combined with a strong acid system, ensuring that all Fe elements can enter the solution without the "lattice residue" or "incomplete digestion" problems common in traditional methods. 3. This method introduces a dual-wavelength absorbance correction model, simultaneously measuring Fe at 510 nm. 2+The method identifies the complexation characteristic peak and the scattering reference peak at 630 nm, and uses an empirically fitted scattering correction coefficient to subtract the absorbance, thereby significantly reducing the signal shift caused by particle scattering. 3. This method effectively reduces the requirements for instruments and environmental conditions, allowing valence state determinations, previously only possible with expensive instruments like the Mössbauer, to be performed on a standard UV-Vis spectrophotometer. The entire process takes less than six hours, making it suitable for routine use in GMP quality control laboratories and directly applicable to the critical quality attribute (CQA) monitoring system of active pharmaceutical ingredients (APIs) or formulations. 4. This method is applicable to various dosage forms, including aqueous phase, oil phase, lyophilized powder, and PEG or SiO2-coated formulations. It has a wide range of applications, high technical accessibility, and provides a practical technical means for the quality control of nano-ferrite drugs. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Fe in the nano-ferrite particles of this invention 2+ and Fe 3+ Spectra before and after color development in one embodiment of a quantitative analysis method for the proportion of oxidation states. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In one embodiment, Fe in nano-ferrite particles is provided 2+ and Fe 3+ A quantitative analysis method for oxidation state ratios was developed. This method, tailored to the complex structural characteristics of nano-ferrite particles, employs a valence state quantitative analysis system that can be performed using a standard UV-Vis spectrophotometer. The overall technical route follows a "cell disruption – protection – digestion – correction – determination" approach. Through steps such as chemical cell disruption, rapid complexation protection, a combination of gentle and complete digestion, and scattering correction, the method achieves the quantitative analysis of Fe... 2+ / Fe 3+ The true measurement.

[0019] Fe in the nano-ferrite particles 2+ and Fe 3+ The quantitative analysis method for oxidation state ratio includes the following steps: (1) Disperse the nano ferrite particle sample in a cell-wall breaking solution for cell-wall breaking treatment.

[0020] In this embodiment, the nano-ferrite sample is dispersed in a cell-breaking solution composed of short-chain thiols and low-concentration hydrofluoric acid. The concentration of short-chain thiols is controlled at approximately 0.08–0.12 mol / L, and the concentration of hydrofluoric acid is controlled at 0.01–0.03 mol / L. The pH of the reaction system is adjusted to approximately 3.5 by adjusting the amounts of short-chain thiols and hydrofluoric acid. After ultrasonic treatment at room temperature for 2–4 minutes, the outer oleic acid layer, PEG layer, or SiO2 shell of the nano-ferrite sample is effectively destroyed, thereby opening the channel for the colorimetric reagent to enter the particle lattice region. Here, the use of low-concentration HF (hydrofluoric acid) is a rigorously validated "narrow window" condition that can break the cell wall without causing Fe 2+ Oxidation.

[0021] In actual measurements, the quantities and ratios of the "nanoferrite sample" and the "short-chain thiol + low-concentration HF cell-wall-breaking solution" are generally fixed. The sample volume of the nanoferrite is approximately 50 mg, and the total volume of the cell-wall-breaking solution is approximately 10 mL (with a short-chain thiol concentration of 0.10 mol / L, an HF concentration of 0.02 mol / L, and a pH adjusted to 3.5). The mass concentration of the sample in the cell-wall-breaking solution is approximately 5 mg / mL. This ratio has been verified through practical operation: it ensures complete submersion and sufficient dispersion of the sample, while preventing local pH / reagent concentration unevenness due to excessively high concentration, which could affect cell-wall breaking and complexation.

[0022] In this method, the mass concentration of the nano-ferrite sample in the cell disruption solution can be controlled within approximately 2–10 mg / mL. Within this concentration range, the cell disruption effect and subsequent measurement results are stable. The corresponding ratio of "sample mass: cell disruption solution volume" can be "1:(100–500) (g:mL)". The ratio is feasible within this range, and the preferred range is approximately 1:(150–300).

[0023] (2) Under inert gas protection conditions, a complexing agent and Fe are added. 2+ Formation of complexes, enabling Fe 2+ It is thus protected from oxidation to Fe during the subsequent acid digestion process. 3+ .

[0024] In this step, the inert gas used can be argon, and the added complexing agent is 2,2'-bipyridine in solid powder form. The bipyridine powder particles have a particle size of 50–100 μm. These bipyridine powder particles (50–100 μm) will dissolve rapidly in the reaction system, allowing Fe to... 2+It can complete the complexation in a very short time (usually less than 30 seconds) to form a highly stable Fe(bipy)3. 2+ Complexes, thereby blocking Fe 2+ It is oxidized to Fe in an acidic environment. 3+ The irreversible process, this instantaneous complexation step is a crucial measure in this method, ensuring that Fe... 2+ The true content is preserved. Additionally, bipyridine is also a colorimetric reagent to support subsequent spectrophotometric determinations.

[0025] (3) The reaction system is placed under relatively mild acidic conditions for preliminary digestion; then, the remaining particles that have not been completely digested are thoroughly digested.

[0026] Here, the system is initially digested under relatively mild acidic conditions by adding 0.1 mol / L hydrochloric acid at a rate of 2-5 mL per 50 mg sample, and maintaining the temperature at approximately 37°C for several minutes. This allows the smaller nano-ferrite particles with more surface defects to dissolve prematurely, further homogenizing the reaction system and reducing the pressure and risk during complete digestion. Following this, the remaining particles that are not completely dissolved need to be thoroughly digested. This is done using an acidic mixture of 1.5 mol / L hydrochloric acid and approximately 0.1 mol / L thioacetic acid, added at a rate of 8-12 mL per 50 mg sample. The molar ratio of HCl to thioacetic acid in the mixed acid is approximately 15:1. The mixture is then heated to approximately 160°C under closed microwave conditions. This combination completely disrupts the nanoparticle lattice, causing the Fe... 2+ with Fe 3+ All of it enters the solution.

[0027] (4) The digested reaction system was used as a colorimetric system, and the absorbance of the colorimetric system at two wavelengths, 510 nm and 630 nm, was measured simultaneously. The 510 nm wavelength is Fe 2+ The wavelength corresponding to the complexation peak, 630 nm, is the wavelength corresponding to the colloidal scattering sensitive region. The scattering intensity at 630 nm is corrected by subtracting the corrected value from the absorbance at 510 nm, yielding the Fe... 2+ True absorbance, through Fe 2+ True absorbance calculation Fe 2+ The concentration.

[0028] Combination Figure 1 As shown, Figure 1 The images show the spectra before and after the color development of bipyridine. After the addition of bipyridine, an absorbance peak is formed at 510 nm, indicating that the addition of bipyridine affects the Fe... 2+ Its antioxidant properties.

[0029] A portion of the reaction system can be used as the colorimetric system. Since the completely digested solution still possesses certain colloidal or subcolloidal scattering properties, Fe at 510 nm can be directly measured using a UV-Vis spectrophotometer. 2+ Complexation absorption peaks are subject to scattering interference, thus requiring spectral correction. This method utilizes simultaneous measurement at 510 nm (Fe) 2+ The absorbance at two wavelengths (complexation peak) and 630 nm (colloidal scattering sensitive region) was used to subtract the scattering contribution to obtain Fe. 2+ The true absorbance corresponding to the complexation absorption peak. The scattering intensity at 630 nm wavelength is corrected using a coefficient of 0.42. This coefficient has been verified to be applicable to common nano-ferrite systems in the 3–20 nm range, ensuring that the corrected absorbance accurately reflects the true Fe. 2+ content.

[0030] Here, the test method for the 0.42 correction factor and the verification experiment showing that this factor is applicable to common nano-ferrite systems in the 3-20nm range are as follows: a) Select solutions of nano-iron oxide particles with different surface modifications (oleic acid and PEG) and different particle sizes (3-5 nm, 5-10 nm, 10-15 nm, 15-20 nm), and determine the true Fe using Mössbauer. 2+ Content C 实 .

[0031] b) After ultrasonic disruption, bipyridine complexation, and microwave digestion of the same batch of sols, the apparent absorbance A at 510 nm was read on a UV-Vis spectrophotometer. 510表 and the scattering signal A at 630nm 630 .

[0032] c) with A 510实 =ε·l·C 实 (ε is Fe(bipy)3 2+ The molar absorptivity is 8650 Lmol. -1 cm -1 (l=1cm) to deduce the theoretical absorbance A under "no scattering" conditions 510实 .

[0033] d) Let ΔA = A 510表 –A 510实 ΔA and the corresponding A 630 Performing linear regression, the slope becomes the scattering correction coefficient k, yielding k = 0.4239, rounded to two decimal places as 0.42. This value corresponds to the Rayleigh scattering "λ" curve. -4 Theoretical estimate (630 / 510) -4 The value ≈0.42 is consistent, indicating that the empirical coefficient has a physical basis. Some experimental data are shown in Table 1 below: Table 1 - Statistical Table of Experimental Data for Correction Coefficient of Colloidal Scattering Intensity at 630nm .

[0034] (5) In Fe 2+ After the concentration is accurately determined, hydroxylamine hydrochloride is added to the same colorimetric system. The concentration of the added hydroxylamine hydrochloride solution is 0.5–2.0 mol / L, preferably about 1.0 mol / L; the volume of the added hydroxylamine hydrochloride solution is 0.5–2.0% (v / v) of the total volume of the colorimetric system, preferably 0.5–1.0% (v / v); the estimated Fe... 3+ The molar ratio of NH₂OH·HCl should be no less than 1:10, preferably 1:50–1:100, to remove Fe from the sample. 3+ Completely reduced to Fe 2+ The absorbance measured again at 510 nm corresponds to Fe 2+ and+Fe 3+ The absorbance of total iron is used to calculate the concentration of total iron. The concentration of total iron is related to Fe. 2+ The concentration difference is Fe 3+ Concentration; ultimately, based on Fe 2+ with Fe 3+ The molar concentration of Fe in nano-ferrite can be used to calculate the Fe content. 2+ / Fe 3+ Oxidation state ratio.

[0035] After measuring the absorbance at 510nm wavelength again, it is necessary to subtract the value corrected for the scattering intensity at 630nm wavelength from the absorbance at 510nm wavelength to obtain the true absorbance of total iron.

[0036] Fe in the nano-ferrite particles 2+ and Fe 3+ The quantitative analysis method for the oxidation state ratio solves the problem of Fe in the existing technology. 2 + Key challenges include easy oxidation, shell-induced color development, difficulty in complete dissolution of nanolattices, colloidal scattering interference with absorbance, and inability to implement QC. This method is applicable to the determination of nanoparticles (containing shells such as PEG / oleic acid / SiO2), can be performed on ordinary UV-Vis analyzers, can complete a batch of sample analysis within 6 hours, and has accuracy comparable to Mössbauer (deviation ≤1.5%). The results can be incorporated into GMP QC methodology.

[0037] Fe in the nano-ferrite particles 2+ and Fe 3+ Quantitative analysis methods for oxidation state ratios can be applied to determine the Fe content in nano-ferrite samples.2+ and Fe 3+ In the oxidation state ratio, the Fe 2+ / Fe 3+ In practical applications, the quantitative analysis method for oxidation state ratio can complete the entire testing process within 6 hours. The only instruments required are a common UV-Vis spectrophotometer and conventional digestion equipment, which can fully meet the conditions of pharmaceutical GMP quality control laboratories. It is suitable for aqueous phase, oil phase, lyophilized powder and various organic / inorganic coated nano ferrite samples.

[0038] Regarding the Fe in these nano-ferrite particles 2+ and Fe 3+ The accuracy of the quantitative analysis method for oxidation state ratio is comparable to that of Mössbauer (deviation ≤ 1.5%), and the verification process for this accuracy is as follows: By conducting a round of methodological comparison experiments, Fe content of nano-ferrite samples from the same batch or source was determined using both this method and Mössbauer spectroscopy. 2+ / Fe 3+ The molar ratio is determined, and then the relative deviations between the two are compared. The specifics are as follows: Three representative sample types were selected: SiO2-coated Fe3O4 lyophilized powder (Fe3O4@SiO2), oleic acid-modified γ-Fe2O3 oil phase dispersion, and PEG (polyethylene glycol)-coated Fe3O4 aqueous phase formulation / lyophilized reconstitution system. At least three batches (or three parallel samples) of each type of sample were prepared, and the Fe content was determined using this method. 2+ / Fe 3+ The proportions were then measured again using Mössbauer on the same sample.

[0039] The calculated relative deviation between "this method vs. Mössbauer" is approximately between 0.5% and 1.3%. A typical example was obtained in a specific measurement instance: this method measured Fe... 2+ / Fe 3+ =0.351, Mössbauer=0.348, relative deviation approximately 0.86%.

[0040] Based on the above experimental results, the performance index of the measurement results in this method is marked as "deviation ≤ 1.5%". This is a conservative upper limit value given after slightly relaxing the maximum deviation of the current actual measurement to leave a safety margin. The actual accuracy may be higher than this.

[0041] The basis for this method's claim of "completing a batch of sample testing within 6 hours" is as follows: The figure of "completing a batch of sample testing within 6 hours" is based on actual operational timing. Following the actual QC workflow, each step from sample weighing to result output was timed with a stopwatch, and the working time for the "same batch of samples" was statistically analyzed. The approximate working time breakdown is as follows: 1. Sample weighing, adding cell wall disrupting solution, adjusting pH, ultrasonic cell wall disruption, adding bipyridine for pre-complexation: approximately 40–60 minutes. 2. Pre-digestion (0.1 mol / L HCl, 37℃) + microwave complete digestion (1.5 mol / L HCl + thioacetic acid, heating, isothermal, cooling): approximately 120–150 minutes. 3. Digestion solution cooling, transfer to volume, necessary dilution: approximately 30 minutes. 4. First colorimetric determination of Fe. 2+ This includes readings at 510nm / 630nm, scattering correction, and data processing. Reduction of Fe with hydroxylamine hydrochloride. 3+ →Fe 2+ The color was developed again, the total iron was measured, and the Fe was calculated. 3+ Fe 2+ / Fe 3+ 5. Instrument warm-up, intermittent waiting, record filling and simple cleanup: approximately 20–30 minutes.

[0042] Adding up all the above steps, the total time is approximately 5.0–5.5 hours. Even considering the small delays that may occur during operation, the overall time can still be stably controlled within 6 hours. Therefore, the statement in this method that "a batch of samples can be tested within 6 hours" is based on actual operation records under QC conditions.

[0043] Fe in the nano-ferrite particles 2+ and Fe 3+ In a specific application example of the quantitative analysis method for oxidation state ratio, this method was used to detect Fe3O4@SiO2 lyophilized powder. The specific detection method and results are as follows: 1. In this embodiment, approximately 50 mg of Fe3O4@SiO2 lyophilized powder was selected as the test sample. The sample was first added to 10 ml of a cell-wall breaking solution composed of short-chain thiols and hydrofluoric acid. The concentration of short-chain thiols was set at approximately 0.10 mol / L, and the concentration of hydrofluoric acid was approximately 0.02 mol / L. The pH of the reaction system was adjusted to approximately 3.5. The sample was then subjected to ultrasonic treatment at room temperature for approximately three minutes. Under these conditions, residual oleic acid, PEG, and the SiO2 shell were effectively destroyed, allowing the iron element inside the particles to be reached by the colorimetric reagent.

[0044] 2. After the cell wall disruption step is completed, approximately 200 mg of 2,2'-bipyridine solid powder is added to the system under argon atmosphere. The powder dissolves rapidly in the solution and reacts with Fe within about thirty seconds. 2+ Complete the complexation, allowing Fe 2+It is thus protected from oxidation during acidic digestion.

[0045] 3. Subsequently, smaller particles were dissolved first by adding 0.1 mol / L hydrochloric acid and maintaining the system at a mild temperature of approximately 37°C for five minutes. Immediately afterwards, the system was transferred to a mixed acid solution consisting of 1.5 mol / L hydrochloric acid and 0.1 mol / L thioacetic acid, and the temperature was raised to approximately 160°C in a closed microwave digestion apparatus to ensure complete digestion of the SiO2-coated particles.

[0046] 4. After digestion, Fe at a wavelength of 510 nm was measured using a UV-Vis spectrophotometer. 2+ The absorption peak was complexed, and the scattering signal at 630 nm was recorded simultaneously. The absorbance was corrected using a scattering correction coefficient of 0.42 determined from previous fitting studies to obtain the true Fe... 2+ Concentration. Hydroxylamine hydrochloride was then added to the same colorimetric solution to adjust the Fe concentration. 3+ The absorbance is completely reduced and measured again; the difference is Fe. 3+ Content. The final calculated Fe content. 2+ / Fe 3+ The ratio is 0.351, and the deviation from 0.348 obtained by Mössbauer is only 0.86%, proving that this method has the same accuracy as high-end equipment.

[0047] In this embodiment, the measured absorbance data are shown in Table 2 below: Table 2 - Freeze-dried Fe3O4@SiO2 powder 2+ / Fe 3+ Absorbance data obtained by proportional detection step λ / nm Absorbance Abs Remark <![CDATA[1.Fe 2+ ]]> 510 0.120 Without hydroxylamine hydrochloride, three averages Scattering reference 630 0.068 Three averages 2. Total iron after adding hydroxylamine hydrochloride 510 0.375 Three averages 3. Blank 510 0.002 The three averages in the table above refer to the average of three measurements.

[0048] Based on the data in Table 2 above, Fe 2+ / Fe 3+ The calculation process for the ratio is as follows: Fe 2+ True absorbance: 0.120 - 0.42 × 0.068 - 0.002 = 0.089; Fe 2+ Concentration (mol / L): 0.089 / 8650 = 1.034 × 10 -5 ; The true absorbance of total iron is: 0.375 - 0.42 × 0.068 = 0.344; Total Fe concentration (mol / L): 0.344 / 8650 = 3.982 × 10⁻⁶ -5 ; Fe3+ Concentration (mol / L): 3.982 × 10 -5 -1.034×10 -5 =2.948×10 -5 ; Fe 2+ / Fe 3+ (Molar ratio): 1.034 × 10 -5 / 2.948×10 -5 =0.351.

[0049] In the above calculation process, 8650 represents Fe(bipy)3. 2+ The molar absorptivity is 0.42; 0.42 is the scattering correction coefficient determined by fitting, used to correct the absorbance.

[0050] The Mössbauer spectroscopy detection process is as follows: The Fe3O4@SiO2 lyophilized powder sample of this embodiment was subjected to... 57 FeMössbauer analysis at 80 K showed that the spectrum could be decomposed into two main hyperfine splitting components. The isomer shift and tetrapolar splitting parameters of one component were related to octahedral coordinated Fe. 2+ Consistent, with a relative area of ​​approximately 25.8%; the other component corresponds to Fe. 3+ The combined contribution (including tetrahedral and octahedral sites) accounts for approximately 74.2% of the relative area. Assuming similar Lamb–Mössbauer factors, this area ratio can approximate the proportion of iron atoms in the corresponding valence states, thus yielding the Fe... 2+ / Fe 3+ The molar ratio is approximately 0.348.

[0051] In contrast, the method used in this embodiment measures Fe at room temperature. 2+ / Fe 3+ The ratio is 0.351, and the relative deviation between the two is approximately 0.86%, indicating that the method used in this embodiment can achieve Fe values ​​comparable to those obtained by the low-temperature Mössbauer test under ordinary analytical laboratory conditions. 2+ / Fe 3+ Quantitative accuracy.

[0052] Fe in the nano-ferrite particles 2+ and Fe 3+ In another specific application example of the quantitative analysis method for oxidation state ratio, the detection method and results of oleic acid modified γ-Fe2O3 oil phase dispersion using this method are as follows: 1. In this example, approximately 0.2 mL of oleic acid-modified γ-Fe₂O₃ colloidal dispersion was selected. Due to the relatively dense oleic acid layer, the concentration of hydrofluoric acid in the cell disruption solution was set to the upper limit of approximately 0.03 mol / L, the thiol concentration was set to 0.12 mol / L, and the ultrasonic treatment time was extended to nearly four minutes to ensure that the coordination structure of the oleic acid layer was fully broken, allowing the subsequent colorimetric agent to enter the interior of the particles.

[0053] 2. After the cell wall was broken, solid bipyridine was added to the sample under argon protection, causing Fe... 2+ The iron ions were quickly complexed and protected, then subjected to mild acid treatment and complete microwave digestion according to standard procedures. After all iron ions had entered the solution, the Fe content was determined by dual-wavelength absorbance measurement and the scattering subtraction formula. 2+ Concentration calculation.

[0054] 3. In this type of oil-phase system, the method can achieve a recovery rate of approximately 98.7% through spiked recovery experiments, with a repeatability accuracy (RSD) of approximately 1.5%, indicating that the method is suitable for oleic acid-coated systems and can maintain good stability and accuracy.

[0055] The spiked recoveries and repeatability of this method in the oleic acid-coated nano-ferrite system are verified as follows: For oleic acid-modified γ-Fe₂O₃ oil phase dispersions, the spike recovery and precision of this method were verified using the "difference method before and after spiking". Six samples from the same batch were taken, and the iron concentration C₀ before spiking was measured to obtain the total Fe mass m₀ in the samples. Then, Fe was quantitatively added to the corresponding samples. 2+ Standard solution, making Fe 2+ The actual amount of spiking is m 标 The concentration C after spiking was determined according to the following procedure: cell disruption – pre-complexation – two-step digestion – dual-wavelength correction – hydroxylamine reduction. 标+ The total Fe mass after spiking was obtained as m. 标+ With Δm=m 标+ -m0 represents the measured spiking amount, as shown in Table 3 below. Table 3 presents the experimental statistics of spiking recovery in the oleic acid-coated nanoferrite system. The calculated average Δm for the six samples was approximately 9.88 μg, corresponding to an average recovery of approximately 98.7% and a relative standard deviation (RSD) of approximately 1.5%. These results indicate that this method still exhibits good spiking recovery and repeatability in the oleic acid-coated nanoferrite system, meeting the requirements for quantitative analysis.

[0056] Table 3 - Statistical data on the recovery rate of oleic acid-coated nanoferrite systems. Serial Number <![CDATA[m0 / μg]]> <![CDATA[m 标 / μg]]> <![CDATA[m 标+ / μg]]> <![CDATA[△ m / μg]]> Recovery rate 1 10.11 10 19.72 9.61 96.1% 2 9.95 10 19.75 9.8 98.0% 3 10.05 10 19.93 9.88 98.8% 4 9.9 10 19.86 9.96 99.6% 5 9.98 10 20.03 10.05 100.5% 6 10.02 10 19.98 9.96 99.6%

[0057] Fe in the nano-ferrite particles 2+ and Fe3+ In another specific application example of the quantitative analysis method for oxidation state ratio, this method was used to detect PEG-coated Fe3O4 lyophilized powder (verified under low intensity conditions). The specific detection method and results are as follows: 1. In this embodiment, 50 mg of PEG-coated lyophilized Fe3O4 powder was reconstituted with 1 mL of water. To verify the adaptability of this method under low reaction intensity, this embodiment used the lowest concentration range of the cell-wall breaking agent: hydrofluoric acid approximately 0.01 mol / L, thiol approximately 0.08 mol / L, ultrasonic treatment time approximately two minutes, and bipyridine dosage of 150 mg. Despite the mild conditions, the PEG shell could still be effectively broken down, allowing the colorimetric agent to penetrate the shell and enter the particle, reacting with the released Fe3O4. 2+ Formation of complexes.

[0058] 2. After completing one mild digestion and one complete digestion, dual-wavelength absorbance measurement can be performed, and accurate Fe values ​​can be obtained according to the scattering correction relationship. 2+ Concentration. Hydroxylamine hydrochloride was then added to obtain the total iron content, and Fe was calculated accordingly. 3+ with Fe 2+ The proportion. For the testing of three parallel batches, the obtained Fe... 2+ / Fe 3+ The proportional repeatability RSD is 1.9%, and the entire test cycle is controlled within approximately 5.6 hours, fully meeting the GMP environment's requirements for the speed and stability of QC methods.

[0059] The verification process for the proportional repeatability RSD in this embodiment is as follows: According to this method, Fe was measured without the addition of hydroxylamine hydrochloride after digestion. 2+ The concentration was determined by adding hydroxylamine hydrochloride to obtain the total Fe concentration, and the difference between the two concentrations was used to obtain the Fe concentration. 3+ Concentration, thus obtaining Fe 2+ / Fe 3+ The ratio, with an RSD of 1.9% for three consecutive tests, is as follows: <![CDATA[Fe 2+ ]]> Total Fe <![CDATA[Fe 3+ ]]> <![CDATA[Fe 2+ / Fe 3+ ]]> 10.24 40.17 29.93 0.342 10.15 38.8 28.65 0.354 10.45 40.91 30.46 0.343

[0060] Based on the above embodiments, it can be seen that this method utilizes a combined system of cell disruption, protection, complete digestion, and spectral correction designed specifically for the characteristics of nano-ferrites to enable Fe... 2+ / Fe 3+The oxidation state ratio can be accurately determined under conventional UV-Vis instrument conditions, achieving analytical precision close to that of Mössbauer spectroscopy. Due to the use of a cell-breaking system composed of low-concentration hydrofluoric acid and short-chain thiols, the oleic acid, PEG, or SiO2 coatings on the outer layer of the nanoparticles can be effectively opened under relatively mild conditions, allowing the chromogenic reagent to penetrate the shell and enter the lattice region, avoiding the "false negative" problem caused by color development only occurring on the surface in traditional methods. The subsequent introduction of solid-state bipyridine enables rapid determination of Fe... 2+ Rapid complexation successfully blocked its oxidation to Fe in acidic systems. 3+ The spontaneous reaction of Fe 2+ It was able to be completely preserved during the analysis. It was verified that Fe... 2+ The oxidation loss is reduced from 10%–15% in traditional methods to about 1%, which significantly improves the reliability of the measurement data.

[0061] Furthermore, the two-step digestion technique employed in this method considers both the initial dissolution characteristics of nanoparticles under low acidity conditions and the complete digestion of various coated nano-ferrites through a closed microwave combined with a strong acid system. This ensures that all Fe elements can enter the solution, avoiding the "lattice residue" or "incomplete digestion" problems commonly found in traditional methods. The digested solution still retains certain colloidal scattering properties, a characteristic long neglected in previous methods, leading to systematic biases in absorbance measurements. This method introduces a dual-wavelength absorbance correction model, simultaneously measuring Fe at 510 nm. 2+ The absorbance was subtracted by combining the complexation characteristic peak with the scattering reference peak at 630 nm and using an empirically fitted scattering correction coefficient, thereby significantly reducing the signal shift caused by particle scattering. Linearity verification showed that the correlation coefficient of the corrected measurement results reached 0.9996, significantly better than traditional single-wavelength measurements.

[0062] Therefore, this method not only significantly improves the accuracy and repeatability of valence state analysis of nano-ferrites, but also effectively reduces the requirements for instruments and environmental conditions, enabling valence state determinations that previously relied on expensive instruments such as Mössbauer to be performed on a common UV-Vis spectrophotometer. The entire process takes less than six hours, making it suitable for routine use in GMP quality control laboratories and directly integrating into the critical quality attribute (CQA) monitoring system for active pharmaceutical ingredients or formulations. This method is applicable to various dosage forms, including aqueous phase, oil phase, lyophilized powder, and PEG or SiO2-coated formulations, offering broad applicability and high technical accessibility, providing a practical and feasible technical means for the quality control of nano-ferrite drugs. Implementation in enterprises can significantly shorten batch release cycles, improve testing efficiency, reduce testing costs, and enhance product consistency and regulatory acceptability, demonstrating outstanding industrial application value and promotional significance.

[0063] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Fe in a type of nano-ferrite particles 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized in that, The method includes: The nano-ferrite particle sample was dispersed in a cell-wall breaking solution for cell-wall breaking treatment. Under inert gas protection conditions, a complexing agent and Fe are added. 2+ Formation of complexes, enabling Fe 2+ It is thus protected from oxidation to Fe during the subsequent acid digestion process. 3+ ; The reaction system was initially digested under relatively mild acidic conditions; subsequently, any remaining particles that were not completely digested were thoroughly digested. The digested reaction system was used as a colorimetric system, and the absorbance of the system at two wavelengths, 510 nm and 630 nm, was measured simultaneously. The 510 nm wavelength represents the absorbance of Fe. 2+ The wavelength corresponding to the complexation peak, 630 nm, is the wavelength corresponding to the colloidal scattering sensitive region. The scattering intensity at 630 nm is corrected by subtracting the corrected value from the absorbance at 510 nm, yielding the Fe... 2+ True absorbance, through Fe 2+ True absorbance calculation Fe 2+ The concentration; In Fe 2+ After the concentration was accurately determined, hydroxylamine hydrochloride was added to the same colorimetric system to remove Fe from the sample. 3+ Completely reduced to Fe 2+ The absorbance measured again at 510 nm corresponds to Fe 2+ and+Fe 3+ The absorbance of total iron is used to calculate the concentration of total iron. The concentration of total iron is related to Fe. 2+ The concentration difference is Fe 3+ Concentration; ultimately, based on Fe 2+ with Fe 3+ The molar concentration of Fe in nano-ferrite can be used to calculate the Fe content. 2+ / Fe 3+ Oxidation state ratio.

2. Fe in the nano-ferrite particles according to claim 1 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized in that, The process of dispersing nano-ferrite particle samples in a cell-wall breaking solution for cell-wall breaking includes: dispersing the nano-ferrite sample in a cell-wall breaking solution composed of short-chain thiols and low-concentration hydrofluoric acid to form a preliminary reaction system, wherein the concentration of the short-chain thiols is controlled at 0.08–0.12 mol / L, the concentration of the hydrofluoric acid is controlled at 0.01–0.03 mol / L, and the pH of the reaction system is adjusted to approximately 3.5; after the sample in the reaction system is ultrasonically treated at room temperature for 2–4 minutes, the outer oleic acid layer, PEG layer, or SiO2 shell will be effectively destroyed, thereby opening the channel for the colorimetric reagent to enter the particle lattice region.

3. Fe in the nano-ferrite particles according to claim 2 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized by: During the cell wall disruption process, the ratio of "sample mass: cell wall disruption solution volume" between the nano-ferrite particle sample and the cell wall disruption solution is "1:(100–500) (g:mL)".

4. Fe in the nano-ferrite particles according to claim 3 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized by: During the cell wall disruption process, the ratio of "sample mass: cell wall disruption solution volume" between the nano-ferrite particle sample and the cell wall disruption solution is "1:(150–300) (g:mL)".

5. Fe in the nano-ferrite particles according to claim 1 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized by: The complexing agent is 2,2'-bipyridine in solid powder form, with a particle size of 50–100 μm, used to make Fe... 2+ The complexation is completed rapidly, forming Fe(bipy)3. 2+ Complex.

6. Fe in the nano-ferrite particles according to claim 1 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized by: The preliminary digestion of the reaction system under relatively mild acidic conditions includes: adding 0.1 mol / L hydrochloric acid to the reaction system and maintaining the temperature at about 37°C for several minutes to dissolve the nano-ferrite particles with smaller particle size and more surface defects in advance. The complete elimination of residual particles that have not been completely eliminated includes: adding an acidic mixture of 1.5 mol / L hydrochloric acid and 0.1 mol / L thioacetic acid to the reaction system, and heating to approximately 160°C under closed microwave conditions to completely destroy the nanoparticle lattice and eliminate the Fe... 2+ with Fe 3+ All of it enters the solution.

7. Fe in the nano-ferrite particles according to claim 6 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized by: The reaction system is to add 0.1 mol / L hydrochloric acid, and 2-5 mL of hydrochloric acid is added for every 50 mg of nano ferrite particle sample. In the acidic mixed system consisting of 1.5 mol / L hydrochloric acid and 0.1 mol / L thioacetic acid added to the reaction system, each 50 mg of nano ferrite particle sample corresponds to 8–12 mL of acidic mixed system, and the molar ratio of hydrochloric acid to thioacetic acid in the acidic mixed system is approximately 15:

1. The concentration of hydroxylamine hydrochloride solution added to the same colorimetric system is 0.5–2.0 mol / L; the volume percentage of the added hydroxylamine hydrochloride is 0.5–2.0% (v / v) of the total volume of the colorimetric system; Fe 3+ The molar ratio with hydroxylamine hydrochloride is not less than 1:

10.

8. Fe in the nano-ferrite particles according to claim 1 2+ and Fe 3+ A quantitative analysis method for oxidation state ratio, characterized by: The scattering intensity at a wavelength of 630 nm was corrected with a coefficient of 0.

42.

9. Fe in the nano-ferrite particles according to any one of claims 1-8 2+ and Fe 3+ A quantitative analysis method for the oxidation state ratio in determining Fe in nano-ferrite samples 2+ and Fe 3+ Application in oxidation state ratios.

10. The application according to claim 9, characterized in that: The nano-ferrite samples are aqueous phase, oil phase, lyophilized powder, and various organic / inorganic coated nano-ferrite samples.

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