A method for detecting L-5-methyltetrahydrofolate content resistant to magnesium ion interference

Abstract

This invention discloses a method for detecting L-5-methyltetrahydrofolate content with magnesium ion interference resistance, comprising the following steps: S1. Sample pretreatment: S11. Using phosphate buffer containing antioxidants, disodium EDTA, and sodium citrate as the extraction reagent, the sample is ultrasonically extracted in an ice-water bath at 2-6°C under light-protected conditions; S12. Extraction is performed using an aqueous two-phase extraction system; S2. HPLC detection: A reversed-phase chromatographic column is used, with gradient elution using mobile phase A and mobile phase B; mobile phase A is an aqueous solution of potassium dihydrogen phosphate containing antioxidants, sodium citrate, and disodium EDTA, and mobile phase B is methanol. This invention solves the problems of peak tailing, signal suppression, and matrix interference in the detection of L-5-methyltetrahydrofolate in complex magnesium-containing systems by synergistically dissociating the magnesium-folate complex through pH control and a composite masking agent, combined with aqueous two-phase extraction to purify the matrix, and optimized HPLC separation conditions.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting L-5-methyltetrahydrofolate content that resists magnesium ion interference. Background Technology

[0002] L-5-methyltetrahydrofolate (5-MTHF), as the active form of folic acid, is widely used in nutritional supplements and pharmaceuticals. In magnesium-containing compound systems (such as multivitamins and prenatal vitamins), magnesium ions (Mg²⁺) are present. 2+ It significantly interferes with the detection of L-5-methyltetrahydrofolate: 1. Complexation leads to separation failure: Mg 2+ It forms a stable complex with the pteroylglutamic acid structure of L-5-methyltetrahydrofolate (stability constant K≈10). 4 This causes chromatographic peaks to tail (tailing factor T > 1.5), or even co-elute with impurity peaks, resulting in a resolution Rs < 1.2.

[0003] 2. Signal suppression affects quantification: Mg 2+ Although there is no direct absorption at the detection wavelength (292 nm), by changing the conjugated structure of L-5-methyltetrahydrofolate, the UV response value is reduced by 10% to 20%, and the spiked recovery rate is only 80% to 85%.

[0004] 3. Matrix complexity exacerbates interference: Vitamin B in compound systems 12 Components such as amino acids and Mg 2+ Synergistic effects lead to the formation of multi-component complexes, which traditional single masking agents (such as EDTA) cannot completely eliminate, and solid-phase extraction (SPE) purification efficiency is less than 60%.

[0005] Existing methods (such as GB 5413.31-2013) do not have specific solutions designed for magnesium ion interference, resulting in detection errors of >15% for magnesium-containing samples. There is an urgent need to develop a specific detection technology resistant to magnesium interference to meet the requirement for accurate quantification of L-5-methyltetrahydrofolate in highly complex matrices. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting L-5-methyltetrahydrofolate content that is resistant to magnesium ion interference, in order to overcome the shortcomings of the prior art.

[0007] The objective of this invention is achieved through the following technical solution: A method for detecting L-5-methyltetrahydrofolate content resistant to magnesium ion interference, comprising the following steps: S1. Sample pretreatment: S11. Using a phosphate buffer containing antioxidants, disodium EDTA, and sodium citrate as the extraction reagent, the sample was ultrasonically extracted in an ice-water bath at 2-6°C under light-protected conditions; the concentrations of sodium citrate and disodium EDTA were 0.10-0.30 mol / L and 0.05-0.15 mol / L, respectively, and the pH of the phosphate buffer was 5.8-6.3; S12. Then, an aqueous two-phase extraction system was used for extraction to obtain the test solution; S2. HPLC detection: The content of L-5-methyltetrahydrofolate in the test solution was detected by HPLC; the chromatographic conditions were as follows: A reversed-phase column was used for gradient elution with mobile phase A and mobile phase B; The mobile phase A is a 0.01-0.03 mol / L aqueous solution of potassium dihydrogen phosphate containing antioxidants, sodium citrate, and disodium EDTA, with a pH of 5.8-6.3; the concentrations of sodium citrate and disodium EDTA are 0.05-0.20 mol / L and 0.01-0.03 mol / L, respectively. The mobile phase B is methanol; The gradient elution procedure is shown in Table 1: Table 1 .

[0008] Preferably, for samples rich in protein and / or fat, the following step is further included between steps S11 and S12: Add 20-50% of the volume of the extract to the extract in step S11, vortex mix, then centrifuge at low temperature, discard the supernatant and proceed to step S12. Preferably, the protein + fat content in the protein- and / or fat-rich sample is >20%.

[0009] Preferably, the antioxidant in steps S1 and S2 is ascorbic acid; The mass percentage concentration of ascorbic acid in the phosphate buffer solution described in step S11 is 0.08~0.12%; The mass percentage concentration of ascorbic acid in the mobile phase A in step S2 is 0.08~0.12%.

[0010] Preferably, the ratio of the sample to the phosphate buffer in step S11 is 0.2~0.6 g / 100 mL.

[0011] Preferably, the ultrasonic extraction frequency in step S11 is 200~500w, and the time is 15~25min.

[0012] Preferably, the extractant used in the aqueous two-phase extraction system is polyethylene glycol and potassium dihydrogen phosphate; The ratio of the amount of polyethylene glycol, the amount of potassium dihydrogen phosphate, and the amount of phosphate buffer solution is 15~25g / 100mL and 10~20g / 100mL, respectively.

[0013] The phosphate buffer solution in step S11 is selected from any one of the following: potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer pair, sodium dihydrogen phosphate and disodium hydrogen phosphate buffer pair.

[0014] Preferably, the chromatographic column is a C18 chromatographic column; The C18 chromatographic column has an inner diameter of 4.6 mm and a length of 150 mm, a packing particle size of 3.5 μm, and a packing pore size of 120 Å.

[0015] Preferably, the detection wavelength in step S2 is 292 nm; the flow rate of the mobile phase in step S2 is 0.8~1.2 mL / min, and the column temperature is 28~32℃.

[0016] 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 ℃, the detection wavelength was 292 nm, and the injection volume was 10 μL. The mobile phase A is a 0.02 mol / L aqueous solution of potassium dihydrogen phosphate containing antioxidants, sodium citrate, and disodium EDTA, with a pH of 5.9; the concentrations of sodium citrate and disodium EDTA are 0.1 mol / L and 0.02 mol / L, respectively. The mobile phase B is methanol; The gradient elution procedure is as follows: During the first 5 minutes, the volume fraction of mobile phase B increased from 10% to 25%, with the remainder being mobile phase A. Between 5 and 10 minutes, the volume fraction of mobile phase B increased from 25% to 35%, with the remainder being mobile phase A. Between 10 and 15 minutes, the volume fraction of mobile phase B decreased from 35% to 10%, with the remainder being mobile phase A. Quantitative analysis was performed using the external standard method.

[0017] This invention provides a method for detecting L-5-methyltetrahydrofolate (L-5-methyltetrahydrofolate) content with resistance to magnesium ion interference. By synergistically dissociating magnesium-folate complexes through pH adjustment and composite masking agents, combined with aqueous two-phase extraction to purify the matrix, and with optimized HPLC separation conditions, the method solves the problems of peak tailing, signal suppression, and matrix interference in the detection of L-5-methyltetrahydrofolate in complex magnesium-containing systems. Attached Figure Description

[0018] Figure 1 This is the chromatogram of the sample from Example 1 obtained by HPLC detection. Detailed Implementation

[0019] This application provides a method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference, comprising the following steps: S1. Sample pretreatment: S11. Using a phosphate buffer containing antioxidants, disodium EDTA, and sodium citrate as the extraction reagent, the sample was extracted by sonication in an ice-water bath at 2-6℃ under light-protected conditions; the concentrations of sodium citrate and disodium EDTA were 0.10-0.30 mol / L and 0.05-0.15 mol / L, respectively, and the pH of the phosphate buffer was 5.8-6.3.

[0020] Using a phosphate buffer solution with a pH of 5.8–6.3 as the extraction reagent has the following advantages: 1. Maintaining the optimal pH environment: Provide a stable, weakly acidic chemical environment (pH 5.8~6.3) for L-5-methyltetrahydrofolate, which is its most stable pH range, and can minimize degradation under acid / base catalysis.

[0021] Limiting the pH of the phosphate buffer is the most critical measure for controlling the chemical form and stability of L-5-methyltetrahydrofolate at its source. L-5-methyltetrahydrofolate is most stable under near-neutral, weakly acidic conditions. Too low a pH (strong acid) or too high a pH (alkaline) will accelerate the ring-opening degradation or isomerization of its pteridine ring. Precisely controlling the pH of the extraction solution between 5.8 and 6.3 minimizes the risk of degradation from the very first step of analysis.

[0022] 2. Provides ionic strength: helps to disrupt the structure of the sample matrix (such as tablet excipients), promoting the release and dissolution of the target analyte from the solid phase.

[0023] 3. Buffering external acid and alkali interference: The sample may contain trace amounts of acidic or alkaline components. The phosphate buffer system can effectively counteract their effects and ensure pH stability during the extraction process.

[0024] 4. Compatibility with subsequent steps: Maintains chemical consistency with subsequent aqueous two-phase extraction (potassium dihydrogen phosphate) and chromatographic mobile phase (phosphate buffer system), reducing errors caused by incompatibility between systems.

[0025] The use of antioxidants and low-temperature ultrasonic extraction in the dark effectively prevents the oxidative degradation of L-5-methyltetrahydrofolate, reducing its degradation rate to <2% / h. Antioxidants such as ascorbic acid are preferred, as they exhibit better antioxidant effects compared to other antioxidants used in this application. The mass percentage concentration of ascorbic acid in the phosphate buffer solution is 0.08~0.12%.

[0026] Phosphate buffer can be a potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer pair, or a sodium dihydrogen phosphate and disodium hydrogen phosphate buffer pair, etc. The concentration of the phosphate buffer is not particularly limited. Preferably, the sample-to-phosphate buffer ratio is 0.2~0.6 g / 100 mL. Within this range, sufficient contact with the sample is achieved, ensuring complete extraction of L-5-methyltetrahydrofolate from the sample.

[0027] Preferably, the ultrasonic extraction frequency is 200~500w and the time is 15~25min.

[0028] S12. Then, an aqueous two-phase extraction system was used for extraction to obtain the test solution; Extraction can be achieved using methods such as oscillation.

[0029] Preferably, the molecular weight of polyethylene glycol is 2000~6000.

[0030] A two-phase system was adopted, utilizing the hydrophilicity of L-5-methyltetrahydrofolate (partition coefficient K=3.2) and Mg... 2+ The salting-out effect enables efficient separation of water-soluble target substances from fat-soluble impurities (such as vitamins A and D), resulting in an extraction recovery rate of >95%, which is superior to traditional C18 column extraction (82%).

[0031] The preferred extractant for the aqueous two-phase extraction system is polyethylene glycol-potassium dihydrogen phosphate, wherein the ratio of the amount of polyethylene glycol and potassium dihydrogen phosphate added to the amount of phosphate buffer is 15~25g / 100mL and 10~20g / 100mL, respectively.

[0032] Polyethylene glycol (PEG) was used as the phase-forming polymer, and potassium dihydrogen phosphate (KH2PO4) was used as the inorganic salt to construct an aqueous two-phase extraction system. This system utilized the salting-out effect of the high-concentration salt to drive the formation of the aqueous two phases and facilitated the interaction of lipophilic impurities with the hydrophilic target analyte (and Mg2+). 2+ The process involves separation of L-5-methyltetrahydrofolate into the inorganic salt phase and lipid-soluble impurities into the polyethylene glycol phase, achieving "liquid-liquid extraction purification".

[0033] The addition of potassium dihydrogen phosphate significantly increases the pH of the entire system. As mentioned earlier, high pH may affect the stability of L-5-methyltetrahydrofolate. However, L-5-methyltetrahydrofolate has already been extracted from the complex matrix and transferred to a relatively pure aqueous phase. The exposure time to an unfavorable pH environment is extremely short, and chromatographic analysis will be performed immediately afterward (the mobile phase pH will return to the optimized 5.8–6.3). Therefore, this brief and controllable pH change will not pose a substantial threat to the stability of L-5-methyltetrahydrofolate.

[0034] As will be understood by those skilled in the art, preferably, step S1, before HPLC detection, further includes a step of passing the aqueous phase through a 0.22 μm microfiltration membrane to prevent impurities in the solution from clogging the chromatographic column.

[0035] The test solution should be analyzed by HPLC as soon as possible. If it needs to be stored for a short period of time, it must be stored at 4°C in the dark to reduce the degradation of L-5-methyltetrahydrofolate.

[0036] Preferably, for samples rich in protein and / or fat, preferably, the protein + fat content in the samples rich in protein and / or fat is >20%, the steps between steps S11 and S12 further include the following steps to fully remove protein and fat from the sample.

[0037] Add 20-50% (by volume) acetonitrile to the extract from step S11, vortex to fully denature and precipitate the protein, then centrifuge at low temperature (2-6°C), and collect the supernatant for step S12. Acetonitrile is used to precipitate the protein in this step because it is miscible with water and has minimal impact on the stability of L-5-methyltetrahydrofolate. Acetonitrile denatures the protein and also induces the precipitation of some lipids by altering the solution polarity. Centrifugation after precipitation effectively removes most of the protein and some lipids, preventing column clogging and detection interference.

[0038] Mechanism of protein removal: precipitation (denaturation); Acetonitrile is a highly polar solvent that can strongly strip the hydration layer on the surface of protein molecules and destroy the hydrogen bonds and other forces in their three-dimensional structure, leading to protein denaturation and aggregation into insoluble precipitates.

[0039] Mechanism of fat (lipid) removal: partitioning and precipitation (alteration of solubility); acetonitrile is miscible with water, and its addition reduces the polarity of the entire solution system. Many lipids (such as triglycerides and phospholipids) have decreased solubility in low-polarity environments, resulting in partial precipitation. Acetonitrile also dissolves some lipids. S2. HPLC detection: HPLC was used to detect the L-5-methyltetrahydrofolate content in the test solution.

[0040] After pretreatment, the test solution contained not only L-5-methyltetrahydrofolate but also some water-soluble impurities. This application uses HPLC to detect the ascorbate palmitate content.

[0041] The chromatographic conditions are as follows: A reversed-phase column was used for gradient elution with mobile phase A and mobile phase B; A C18 column is preferred for reversed-phase chromatography. L-5-methyltetrahydrofolate is a moderately polar molecule with a glutamate moiety (hydrophilic) and a pterin ring moiety (hydrophobic). The C18 stationary phase is a classic medium-to-long-chain alkyl-bonded phase that provides moderate hydrophobic interactions for this molecule, which has both polar and nonpolar structures, thereby achieving good retention and separation.

[0042] The preferred specifications are an inner diameter of 4.6 mm × a length of 150 mm, a packing particle size of 3.5 μm, and a packing pore size of 120 Å. Using a short column can shorten the analysis time (total run time ≤ 15 min), while optimizing the pore size ensures column efficiency and sample loading, and enhances the retention of L-5-methyltetrahydrofolate (retention time tR = 7.5 min).

[0043] As will be understood by those skilled in the art, the detection wavelength is the maximum absorption wavelength of L-5-methyltetrahydrofolate, which is 292 nm.

[0044] Mobile phase A is a 0.01–0.03 mol / L aqueous solution of potassium dihydrogen phosphate containing antioxidants, sodium citrate, and disodium EDTA, with a pH of 5.8–6.3, which can be adjusted by phosphoric acid; the concentrations of sodium citrate and disodium EDTA are 0.05–0.20 mol / L and 0.01–0.03 mol / L, respectively. Mobile phase B is methanol; preferably, chromatographically pure methanol is used.

[0045] The mobile phase pH was set to 5.8–6.3 (preferably 5.9 ± 0.1). Based on the Nernst equation and complexation equilibrium theory, this range allows for the partial dissociation of the γ-carboxyl group (pKa ≈ 5.6) of L-5-methyltetrahydrofolate into COO⁻, weakening its interaction with Mg. 2+ Its coordination ability makes the degree of dissociation of the complex >95%.

[0046] In this application, EDTA disodium and sodium citrate are combined as a composite masking agent in the phosphate buffer extractant and mobile phase. EDTA disodium can preferentially chelate Fe. 3+ Cu 2+ Transition metal ions, sodium citrate can react with Mg via hydroxyl groups. 2+ Formation of a six-coordinate chelate (stability constant K≈10) 6Competitive acquisition of complexed Mg 2+ Thus, it specifically captures Mg 2+ , making free Mg 2+ Concentration reduced to 10 -5 Below mol / L, a dual mechanism of "broad-spectrum chelation + targeted scavenging" is formed. Furthermore, experimental optimization shows that when the ratio of disodium EDTA to sodium citrate is approximately 1:5~6 (molar ratio), Mg... 2+ The removal rate reached 98.7%, and the peak area of ​​L-5-methyltetrahydrofolate had an RSD of <1.0% (n=6), which is 50% higher than that of EDTA alone. The specific combination and dosage ratio of "disodium EDTA and sodium citrate" can effectively and synergistically solve the magnesium interference problem.

[0047] The concentration of the composite masking agent in the phosphate buffer extractant is higher than that in the mobile phase to cope with potentially higher levels of Mg in the sample matrix. 2+ load.

[0048] The gradient elution procedure is as follows: In the first stage, the volume fraction of mobile phase B in the mobile phase increases from 8-12% to 22-28%, with the remainder being mobile phase A; the duration of the first stage is 3-7 minutes; the main purpose of this stage is initial elution and preliminary enrichment of the target analyte. In the second stage, the volume fraction of mobile phase B increases from 22-28% to 32-38%, with the remainder being mobile phase A; the duration of the second stage is 3-7 minutes; the main purpose of this stage is core separation and target elution. In the third stage, the volume fraction of mobile phase B decreases from 32-38% to 8-12%, with the remainder being mobile phase A; the second stage lasts 3-7 minutes. The main purpose of this stage is column regeneration and system equilibration.

[0049] Preferably, the antioxidant in mobile phase A is also ascorbic acid, with a mass percentage concentration of 0.08~0.12%, which can effectively prevent the oxidation of L-5-methyltetrahydrofolate during the detection process.

[0050] Preferably, the mobile phase flow rate is 0.8~1.2 mL / min, which can balance column pressure and retention time. The column temperature is selected at 28~32℃, which can reduce the influence of column temperature fluctuation on retention time and make RSD < 0.5%.

[0051] Therefore, this application utilizes a phosphate buffer-methanol mobile phase system with pH 5.8–6.3, combined with gradient elution, to achieve the dissociation and separation of L-5-methyltetrahydrofolate and the magnesium complex.

[0052] In a preferred embodiment, the chromatographic conditions for step S2 are as follows: 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 ℃, the detection wavelength was 292 nm, and the injection volume was 10 μL. Mobile phase A is a 0.02 mol / L aqueous solution of potassium dihydrogen phosphate containing antioxidants, sodium citrate, and disodium EDTA, with a pH of 5.9; the concentrations of sodium citrate and disodium EDTA are 0.1 mol / L and 0.02 mol / L, respectively. Mobile phase B is methanol; The gradient elution procedure is as follows: During the first 5 minutes, the volume fraction of mobile phase B increased from 10% to 25%, with the remainder being mobile phase A. Between 5 and 10 minutes, the volume fraction of mobile phase B increased from 25% to 35%, with the remainder being mobile phase A. Between 10 and 15 minutes, the volume fraction of mobile phase B decreased from 35% to 10%, with the remainder being mobile phase A.

[0053] Injecting 10 μL of the sample under the above chromatographic conditions, the retention time of the L-5-methyltetrahydrofolate peak was 7.5 min, and the resolution with the adjacent impurity peak was Rs = 2.3. Mg 2+ Interference peaks have been completely eliminated.

[0054] Quantitative analysis was performed using the external standard method, which is simple to operate and calculate.

[0055] As those skilled in the art will understand, the external standard method also includes the step of preparing a standard curve using L-5-methyltetrahydrofolate standard under the same detection conditions. The standard curve is used to perform linear regression analysis with concentration as the abscissa and peak area as the ordinate to obtain a linear regression equation. Then, the sample detection results are substituted into the linear regression equation to calculate the content of L-5-methyltetrahydrofolate in the sample.

[0056] Furthermore, as is commonly known in the art, while synthetic L-5-methyltetrahydrofolate exhibits high activity, its stability is extremely poor. Calcium L-5-methyltetrahydrofolate (L-5-MTHFCa) is the calcium salt form of L-5-methyltetrahydrofolate, possessing a stable crystal form. Synthetic L-5-methyltetrahydrofolate is often prepared as calcium L-5-methyltetrahydrofolate to improve its stability. In vivo or during detection, calcium L-5-methyltetrahydrofolate dissociates to release L-5-methyltetrahydrofolate. Therefore, the detection method of this application is also applicable to the detection of samples present in the form of calcium L-5-methyltetrahydrofolate. The equivalent L-5-methyltetrahydrofolate content measured by this method can be converted and reported as the content of calcium L-5-methyltetrahydrofolate in the sample based on its stoichiometric relationship with the calcium salt.

[0057] Furthermore, L-5-methyltetrahydrofolate calcium can be used for quantitative analysis as the standard material for preparing the standard curve. When using L-5-methyltetrahydrofolate calcium as a standard, the sample weight is converted into an equivalent mass of L-5-methyltetrahydrofolate based on the purity and molecular weight indicated on the standard certificate, and this equivalent mass is used as the basis for calculating the concentration of the standard solution and plotting the standard curve.

[0058] Validation of detection methods

[0059] 1. Linearity and Sensitivity Linear range: 0.1~100 μg / mL. The fitted equation for peak area (A) against concentration (C, μg / mL) is: A = 8524.6C + 18.7, R0 2 =0.9998, covering the actual sample concentration (5~80μg / mL).

[0060] Limit of detection (LOD): 0.05 μg / mL (S / N=3), Limit of quantitation (LOQ): 0.1 μg / mL (S / N=10). The sensitivity is twice that of the GB method.

[0061] 2. Anti-interference capability verification (1) Magnesium ion tolerance concentration: Add 0~500ppm Mg to the sample 2+ When the concentration of the composite masking agent in the mobile phase was 0.02 mol / L disodium EDTA + 0.12 mol / L sodium citrate, the recovery rate of L-5-methyltetrahydrofolate remained stable at 98%–102% (as shown in Table 2). However, when using disodium EDTA as a single masking agent, the recovery rate in Mg... 2+ When the concentration is >100 ppm, the recovery rate drops to below 90% (as shown in Table 3).

[0062] (2) Matrix compatibility: For the compound nutritional tablets containing 10 vitamins and 5 minerals, the resolution between L-5-methyltetrahydrofolate and the adjacent peak was Rs=2.1, and the interference rate of impurity peaks was <0.5%.

[0063] Table 2. Effects of L-5-methyltetrahydrofolate on different concentrations of Mg in the composite masking agent system. 2+ Spiked recovery rate

[0064] Table 3. Spiking recoveries of L-5-methyltetrahydrofolate under single EDTA masking agent

[0065] 3. Stability and Durability (1) Solution stability: The peak area RSD of the sample solution was 1.3% after being placed at 4℃ in the dark for 48 hours; the degradation rate was <3% after being placed at room temperature for 24 hours, which is better than the traditional method (degradation rate >10% after 12 hours at room temperature).

[0066] (2) Column life: After 300 consecutive injections, the column efficiency decreased by <15%, and the tailing factor T=1.1 (initial T=1.05), which meets the requirements of industrial-grade detection.

[0067] Compared with the prior art, this application has the following advantages: 1. Dual elimination mechanism for magnesium ions: Through precise pH control and synergistic effect with composite masking agents, magnesium ion elimination is achieved for the first time. 2+ The entire path from the complexed state to the free state is cleared, which solves the shortcomings of the single masking method in traditional methods.

[0068] 2. Green and efficient purification technology: The introduction of a two-phase aqueous extraction system to replace organic solvents improves extraction efficiency by 15% and reduces organic reagent consumption by 50%, which is in line with the concept of green analytical chemistry.

[0069] 3. Dynamic stability control: The entire process of antioxidant design, from sample pretreatment (low temperature and light protection) to mobile phase (antioxidant), controls the degradation rate of L-5-methyltetrahydrofolate to within 3%, ensuring the reliability of sample detection.

[0070] Example 1: Determination of folic acid content in compound nutritional tablets 1. Preparation and determination of standard curve solutions Accurately weigh an appropriate amount of L-5-methyltetrahydrofolate calcium (5-MTHF-Ca) standard (e.g., 10.0 mg, based on 100% purity) and dissolve and dilute to volume with phosphate buffer (pH 5.9) containing 0.1% ascorbic acid, 0.20 mol / L sodium citrate, and 0.1 mol / L disodium EDTA to prepare an L-5-methyltetrahydrofolate standard stock solution with a concentration of approximately 1.0 mg / mL. Store at -20°C protected from light. The phosphate buffer is a 0.1 mol / L potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer pair solution.

[0071] Before use, serially dilute the standard stock solution with the same phosphate buffer (pH 5.9) to prepare a series of standard working solutions of varying concentrations. The recommended concentration gradient is: 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0 μg / mL, covering the expected detection range of the method.

[0072] 2. Chromatographic analysis The above series of standard working solutions were injected sequentially under the chromatographic conditions described in this invention, with each concentration point injected in parallel 2-3 times. The peak area of ​​L-5-methyltetrahydrofolate was recorded.

[0073] The standard solution was analyzed by HPLC under the following chromatographic conditions.

[0074] A C18 column with an inner diameter of 4.6 mm and a length of 150 mm was used, with a packing particle size of 3.5 μm and a pore size of 120 Å. Gradient elution was performed using mobile phases A and B at a flow rate of 1 mL / min, a column temperature of 30 °C, and a detection wavelength of 292 nm.

[0075] Mobile phase A is a 0.02 mol / L aqueous solution of potassium dihydrogen phosphate containing 0.1% ascorbic acid, sodium citrate and disodium EDTA, with a pH of 5.9; the concentrations of sodium citrate and disodium EDTA are 0.1 mol / L and 0.02 mol / L, respectively. Mobile phase B is methanol; The gradient elution procedure is as follows: During the first 5 minutes, the volume fraction of mobile phase B increased from 10% to 25%, with the remainder being mobile phase A. Between 5 and 10 minutes, the volume fraction of mobile phase B increased from 25% to 35%, with the remainder being mobile phase A. Between 10 and 15 minutes, the volume fraction of mobile phase B decreased from 35% to 10%, with the remainder being mobile phase A.

[0076] Inject 10 μL of the sample under the above chromatographic conditions; the retention time of the L-5-methyltetrahydrofolate peak is 7.5 min.

[0077] The equation fitted to peak area (A) against concentration (C, μg / mL) is: A = 8524.6C + 18.7, R0 2 =0.9998.

[0078] 2. Sample pretreatment Weigh 0.2g of sample (compound nutritional tablets, containing Mg) 2+ Add 200 ppm of phosphate buffer (pH 5.9) containing 0.1% ascorbic acid, 0.20 mol / L sodium citrate, and 0.1 mol / L disodium EDTA. Extract by sonication at 4°C in the dark for 20 min, then transfer to a 50 mL centrifuge tube. The phosphate buffer is a 0.1 mol / L potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer pair.

[0079] Add 10g PEG 4000 and 8g KH2PO4, shake to separate the layers, take the aqueous phase and filter it through a 0.22μm filter membrane, and use the filtrate as the test solution.

[0080] 3. Chromatographic analysis Inject 10 μL of the test solution under the chromatographic conditions described above. The retention time of the L-5-methyltetrahydrofolate peak was 7.5 min, and the resolution Rs = 2.3 with the adjacent impurity peak. Mg 2+ Interference peaks were completely eliminated, and the chromatogram is as follows: Figure 1 As shown.

[0081] 4. Content Calculation and Spike Recovery Validation (1) Calculation of sample content The peak area of ​​L-5-methyltetrahydrofolate obtained from the test sample solution was substituted into the standard curve equation (A = 8524.6C + 18.7) plotted in step 1 to calculate the concentration of L-5-methyltetrahydrofolate in the test sample solution (C_sample, μg / mL). Based on the sample weight, dilution factor, and other pretreatment parameters, the content of L-5-methyltetrahydrofolate in the original sample was calculated to be 0.49 mg / g (the product label value is 0.50 mg / g).

[0082] (2) Method for determining spiked recovery To verify the accuracy of this method in real matrices, a spiked recovery method was used for validation. The specific steps are as follows: a. Background sample determination: Accurately weigh a sample of known content to be tested (from the same batch as the aforementioned sample), and follow the steps of "2. Sample pretreatment" and "3. Chromatographic analysis" to determine the background value of L-5-methyltetrahydrofolate in the sample.

[0083] b. Preparation and determination of spiked samples: Accurately weigh another equal volume of the same sample, and quantitatively add a known amount of L-5-methyltetrahydrofolate standard solution to it before dissolving the sample. Subsequently, perform extraction, purification, and chromatographic analysis on this spiked sample and the unspecified sample simultaneously, and determine the total L-5-methyltetrahydrofolate content in the spiked sample.

[0084] c. Recovery rate calculation: Calculate the spiked recovery rate using the following formula: Recovery rate (%) = [(Total measured value - Background value) / Amount added] × 100% The amount added is the absolute amount of L-5-methyltetrahydrofolate in the added standard.

[0085] (3) Verification results In this embodiment, L-5-methyltetrahydrofolate standard equivalent to approximately 100% of its background concentration (i.e., approximately 100 μg) was added to a 0.2 g sample, and the experiment was performed in triplicate (n=3). The average recovery rate was calculated to be 99.2% and the relative standard deviation (RSD) was 1.1% using the method described above. These results indicate that the method of the present invention is effective in the presence of 200 ppm Mg. 2+ It exhibits excellent accuracy and precision in actual sample matrices.

[0086] (4) Comparison with the indicated value Furthermore, the measured L-5-methyltetrahydrofolate content in the sample obtained by this method was compared with the value indicated on the product label, and the deviation was less than 2%, further demonstrating the reliability and applicability of this method in practical applications.

[0087] Example 2: Stability Study of L-5-Methyltetrahydrofolate 1. Accelerated Degradation Experiment Preparation of Mg 2+ The 300 ppm sample solution was placed at 25°C in the dark, 25°C under light, and 4°C in the dark, respectively, and injected for analysis every 12 hours according to the chromatographic analysis conditions of Example 1.

[0088] The results showed that the degradation rate of the 4℃ dark-protected group was 1.8% after 48 hours, the degradation rate of the 25℃ dark-protected group was 5.2% after 24 hours, and the degradation rate of the light-protected group was 15.3% after 12 hours, which verified the necessity of low-temperature dark-protected treatment.

[0089] Example 3: Verification of Method Accuracy

[0090] 1. Magnesium ion tolerance concentration verification Sample preparation: Select a blank matrix powder containing a compound nutrient that does not contain L-5-methyltetrahydrofolate or whose background value is known. Divide it evenly into several portions.

[0091] Magnesium ion addition: Different concentrations of magnesium chloride (MgCl2·6H2O) standard solution were added to each matrix sample to increase the magnesium ion concentration (MgCl2·6H2O) in the final sample. 2+ The concentrations were 0, 50, 100, 200, 300, 400, and 500 ppm. After thorough mixing and low-temperature drying, a series of Mg... 2+ Spiked sample.

[0092] Spiking and determination: Add Mg to each of the above samples 2+ In the spiked samples, a known amount of L-5-methyltetrahydrofolate standard was precisely added. Subsequently, samples at each concentration point were pretreated and analyzed by HPLC according to the complete method described in Example 1. Each concentration level was determined in triplicate.

[0093] Results: As shown in Table 4, in Mg 2+ At concentrations as high as 500 ppm, the spiked recoveries of L-5-methyltetrahydrofolate remained consistently between 98% and 102%, with relative standard deviations (RSDs) all less than 1.5%. This fully demonstrates the excellent tolerance of the method of this invention to interference from high concentrations of magnesium ions.

[0094] Table 4. Different Mg 2+ Spiked recoveries of L-5-methyltetrahydrofolate at the specified concentration (n=3)

[0095] 2. Matrix compatibility Test sample: A commercially available multivitamin and mineral tablet Contains 10 vitamins: Vitamin A (acetate), Vitamin D3, Vitamin E (dl-α-tocopherol acetate), Vitamin B1 (thiamine hydrochloride), Vitamin B2 (riboflavin), Vitamin B6 (pyridoxine hydrochloride), nicotinamide, pantothenic acid, and Vitamin B1. 12 (Cyanocobalamin), Biotin.

[0096] Minerals (4 types): calcium carbonate, ferrous fumarate, zinc gluconate, and selenomethionine.

[0097] The above-mentioned compound nutritional tablets containing 10 vitamins and 4 minerals were processed and analyzed by HPLC according to the method in Example 1. The resolution between L-5-methyltetrahydrofolate and the adjacent peak was Rs=2.1, and the interference rate of impurity peaks was <0.5%.

[0098] Example 4: Determination of L-5-methyltetrahydrofolate in high-protein compound nutritional powder 1. Sample Weighing and Extraction: Accurately weigh 0.1 g (to a minimum of 0.0001 g) of the sample powder into a 50 mL centrifuge tube. Add 20 mL of a 0.1 mol / L potassium dihydrogen phosphate-dipoxat phosphate buffer (pH 5.9) containing 0.1% ascorbic acid and a composite masking agent (0.10 mol / L disodium EDTA + 0.20 mol / L sodium citrate). Vortex to fully disperse the sample.

[0099] 2. Protein precipitation and purification: To remove a large amount of protein, add 5 mL of acetonitrile to the suspension from the previous step, vortex vigorously for 2 minutes, and then centrifuge at 4°C and 10,000 rpm for 10 minutes. Carefully aspirate the supernatant and transfer it to another clean centrifuge tube.

[0100] 3. Aqueous two-phase extraction and purification: Add 4.0 g of polyethylene glycol 4000 (PEG 4000) and 3.0 g of potassium dihydrogen phosphate (KH2PO4) to the supernatant. Shake vigorously for 5 minutes to fully mix and form an aqueous two-phase system, then let stand until completely separated.

[0101] 4. Collection and filtration: Accurately pipette approximately 2 mL of the lower aqueous phase and pass it through a 0.22 μm aqueous microporous membrane. The filtrate is used as the test solution.

[0102] The content was calculated based on the standard curve of Example 1, specifically 0.74 mg / g (labeled value 0.67 mg / g).

[0103] 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 detecting L-5-methyltetrahydrofolate content resistant to magnesium ion interference, characterized in that, Includes the following steps: S1. Sample pretreatment: S11. Using a phosphate buffer containing antioxidants, disodium EDTA, and sodium citrate as the extraction reagent, the sample was ultrasonically extracted in an ice-water bath at 2-6°C under light-protected conditions; the concentrations of sodium citrate and disodium EDTA were 0.10-0.30 mol / L and 0.05-0.15 mol / L, respectively, and the pH of the phosphate buffer was 5.8-6.3; S12. Then, an aqueous two-phase extraction system was used for extraction to obtain the test solution; S2. HPLC detection: The content of L-5-methyltetrahydrofolate in the test solution was detected by HPLC; the chromatographic conditions were as follows: A reversed-phase column was used for gradient elution with mobile phase A and mobile phase B. The mobile phase A is a 0.01-0.03 mol / L aqueous solution of potassium dihydrogen phosphate containing antioxidants, sodium citrate, and disodium EDTA, with a pH of 5.8-6.3; the concentrations of sodium citrate and disodium EDTA are 0.05-0.20 mol / L and 0.01-0.03 mol / L, respectively. The mobile phase B is methanol; The gradient elution procedure is shown in Table 1: Table 1 2. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, For samples rich in protein and / or fat, the following steps are also included between steps S11 and S12: Add 20-50% of the volume of the extract to the extract in step S11, vortex mix, then centrifuge at low temperature, discard the supernatant and proceed to step S12. Preferably, the protein + fat content in the protein- and / or fat-rich sample is >20%.

3. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, The antioxidant mentioned in steps S1 and S2 is ascorbic acid; The mass percentage concentration of ascorbic acid in the phosphate buffer solution described in step S11 is 0.08~0.12%; The mass percentage concentration of ascorbic acid in the mobile phase A in step S2 is 0.08~0.12%.

4. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, The ratio of the sample to the phosphate buffer solution used in step S11 is 0.2~0.6 g / 100 mL.

5. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, The ultrasonic extraction frequency in step S11 is 200~500w, and the time is 15~25min.

6. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, The extractant used in the aqueous two-phase extraction system is polyethylene glycol and potassium dihydrogen phosphate; The ratio of the amount of polyethylene glycol, the amount of potassium dihydrogen phosphate, and the amount of phosphate buffer solution is 15~25g / 100mL and 10~20g / 100mL, respectively.

7. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, The phosphate buffer solution in step S11 is selected from any one of the following: potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer pair, sodium dihydrogen phosphate and disodium hydrogen phosphate buffer pair.

8. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, The chromatographic column is a C18 column; The C18 chromatographic column has an inner diameter of 4.6 mm and a length of 150 mm, a packing particle size of 3.5 μm, and a packing pore size of 120 Å.

9. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference as described in claim 1, characterized in that, The detection wavelength in step S2 is 292 nm; the flow rate of the mobile phase in step S2 is 0.8~1.2 mL / min, and the column temperature is 28~32℃.

10. The method for detecting L-5-methyltetrahydrofolate content with resistance to magnesium ion interference 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 ℃, the detection wavelength was 292 nm, and the injection volume was 10 μL. The mobile phase A is a 0.02 mol / L aqueous solution of potassium dihydrogen phosphate containing antioxidants, sodium citrate, and disodium EDTA, with a pH of 5.9; the concentrations of sodium citrate and disodium EDTA are 0.1 mol / L and 0.02 mol / L, respectively. The mobile phase B is methanol; The gradient elution procedure is as follows: In the first 5 minutes, the volume fraction of mobile phase B increased from 10% to 25%, with the remainder being mobile phase A. Between 5 and 10 minutes, the volume fraction of mobile phase B increased from 25% to 35%, with the remainder being mobile phase A. Between 10 and 15 minutes, the volume fraction of mobile phase B decreased from 35% to 10%, with the remainder being mobile phase A. Quantitative analysis was performed using the external standard method.

Images