Ion chromatography detection method for acetic acid, nitric acid and nitrous acid in culture medium

Abstract

The invention relates to the technical field of analytical chemistry, in particular to an ion chromatography method for separation and detection of acetate, nitrite and nitrate ions in a complex matrix culture medium. The method mainly solves the problems of insufficient separation degree, serious matrix interference, poor detection precision and reproducibility and the like when the conventional ion chromatography technology is used for detecting a complex matrix culture medium.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to ion chromatography detection technology. Specifically, it is a chromatographic method for the efficient and high-precision separation and quantitative detection of acetate, nitrate and nitrite ions in complex matrix culture media. Background Technology

[0002] In biopharmaceuticals, cell culture, and microbial fermentation, culture media are the core substances supporting cell growth, proliferation, and metabolism. Their chemical composition is complex, typically containing various organic acids (such as acetic acid, lactic acid, and succinic acid), inorganic ions (such as chloride ions, nitrate ions, nitrite ions, sulfate ions, and phosphate ions), sugars, amino acids, proteins, and peptides. Acetic acid, as a key metabolic byproduct, can inhibit cell growth through accumulation; while nitrate and nitrite, as nitrogen sources or potential impurities, directly affect cellular metabolic pathways and product synthesis due to changes in their concentrations. Therefore, establishing an analytical method that can simultaneously, rapidly, and accurately monitor the concentrations of acetic acid, nitrate, and nitrite in culture media is crucial for process optimization and quality control.

[0003] Ion chromatography, especially suppressed conductivity detection ion chromatography, has become a routine technique for the analysis of inorganic anions and organic acids due to its high sensitivity, high selectivity, and ability to analyze multiple components simultaneously. However, directly applying general ion chromatography methods to complex culture medium samples faces several prominent technical bottlenecks:

[0004] First, the chromatographic separation between key analytes is insufficient. Small-molecule organic acids such as acetic acid and lactic acid have similar physicochemical properties and exhibit similar retention behaviors under conventional chromatographic conditions, making co-elution highly likely and preventing accurate quantification of each component. Simultaneously, nitrate ( ) and nitrite ( (I) and (II) are a pair of ions that are difficult to separate. When the stationary phase is not properly selected or the elution program is not optimal, their chromatographic peaks often overlap significantly, especially at low concentration levels, where the mutual interference is more pronounced.

[0005] Secondly, severe matrix interference affects the accuracy and stability of detection. High concentrations of proteins, hydrophobic macromolecules, pigments, and salts in the culture medium not only easily clog and contaminate the chromatographic column, leading to a rapid decline in column efficiency and a shortened column life, but also cause significant matrix effects. These effects manifest as suppression or enhancement of the target analyte response signal, baseline drift and fluctuation, and the appearance of ghost peaks, ultimately resulting in reduced method sensitivity, distorted quantitative results, and poor reproducibility.

[0006] To address these challenges, existing technologies typically employ two strategies: one is to perform complex sample pretreatment, such as protein precipitation, ultrafiltration, or solid-phase extraction, to remove matrix interferences; the other is to optimize chromatographic conditions, such as using multidimensional chromatography. However, the former is often cumbersome and time-consuming, and may introduce the loss or contamination of target analytes; the latter requires extensive experience in method development and makes it difficult to perfectly achieve baseline separation and accurate quantification of acetic acid, nitrate, and nitrite under a single chromatographic condition while ensuring analytical efficiency.

[0007] Therefore, there is an urgent need in this field to develop an ion chromatography detection method specifically for complex matrix culture media. This method should effectively overcome matrix interference and achieve high-sensitivity, high-precision simultaneous detection of acetate, nitrite, and nitrate ions under efficient and robust chromatographic separation conditions, in order to meet the urgent need for rapid, real-time monitoring of key chemical components in modern bioprocesses. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an ion chromatography detection method specifically for culture medium samples. This method solves the problems of low detection accuracy and poor reproducibility of acetic acid, nitrate, and nitrite in complex matrices due to insufficient separation and matrix interference, and achieves simultaneous, rapid, and accurate quantification of the three under single chromatographic conditions.

[0009] According to one aspect of the present invention, an ion chromatography method for the separation and detection of acetate, nitrite and nitrate ions in complex matrix culture media is provided, comprising the following steps: (1) Sample pretreatment: the culture medium sample is treated by passing it through a hydrophilic-lipophilic balanced reversed-phase solid-phase extraction column, and the filtrate is collected as the test solution; (2) Chromatographic separation: the test solution obtained in step (1) is injected into an ion chromatography system, a high-capacity hydrophilic anion exchange column is used, and potassium hydroxide solution is used as the eluent for gradient elution; (3) Detection and analysis: the separated ions are detected by a conductivity detector and quantitatively analyzed by a standard curve method.

[0010] Preferably, the packing material of the high-capacity hydrophilic anion exchange column is polystyrene-divinylphenyl with a crosslinking degree of 55%, a particle size of 7 μm, and a theoretical plate number of not less than 7000.

[0011] Preferably, the anion exchange column is a PrinCen PAS-19 column or an equivalent column. The term "equivalent column" as used herein refers to an ion exchange column with the same or similar packing matrix, similar degree of cross-linking, and similar particle size.

[0012] Preferably, the hydrophilic-lipophilic balanced reversed-phase solid-phase extraction column is an Oasis HLB column or an equivalent column; the pretreatment steps include: first activating the column with methanol, then rinsing and equilibrating with ultrapure water, and finally loading the culture medium sample, discarding the first part of the filtrate, and collecting the subsequent filtrate. An equivalent solid-phase extraction column refers to a solid-phase extraction column with similar hydrophilic-lipophilic balanced polymer packing material.

[0013] Preferably, the amount of methanol used for activation is 3-5 mL, the amount of ultrapure water used for balancing is 10-15 mL, and the volume of the discarded first part of the filtrate is 2-3 mL.

[0014] Preferably, the potassium hydroxide rinsing solution is generated online or prepared in advance from high-purity potassium hydroxide concentrate.

[0015] Preferably, the operating parameters of the ion chromatography system include: flow rate: 0.9 - 1.1 mL / min; column temperature: 30 - 35 ℃; injection volume: 20 - 30 μL; suppressor current: 150 - 170 mA; detector cell temperature: 33 - 37 ℃.

[0016] Preferably, the operating parameters are: flow rate: 1.000 mL / min; column temperature: 32.00 ℃; injection volume: 25 μL; suppressor current: 160 mA; detector cell temperature: 35.000 ℃.

[0017] Preferably, the gradient elution program is as follows: 0.0 - 15.0 min, eluent concentration is 3 mmol / L; 15.1 - 25.0 min, eluent concentration linearly increases from 3 mmol / L to 20 mmol / L; 25.1 - 30.0 min, eluent concentration linearly increases from 20 mmol / L to 45 mmol / L; 30.1 - 35.0 min, eluent concentration decreases to 3 mmol / L and remains at equilibrium.

[0018] According to another aspect of the present invention, the application of the above-described ion chromatography method in the quality control of biopharmaceutical, cell culture or microbial fermentation processes is provided for simultaneously monitoring the concentrations of acetate, nitrite and nitrate ions in the culture medium.

[0019] This invention provides a chromatographic detection method specifically for culture medium samples, which solves the problems of insufficient separation and low detection accuracy of acetic acid, nitric acid, and nitrite in complex matrices. Attached Figure Description

[0020] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0021] Figure 1 The chromatogram (optimized conditions) for detecting acetate, nitrite and nitrate ions in a 2 ppm standard sample according to Example 2 of the present invention.

[0022] Figure 2 The chromatogram is for the detection of acetate, nitrite and nitrate ions in a 2 ppm standard sample according to Comparative Example 1.

[0023] Figure 3 The chromatogram is for the detection of acetate, nitrite and nitrate ions in a 2 ppm standard sample according to Comparative Example 2.

[0024] Figure 4 The chromatograms of the culture medium sample provided according to the present invention and the culture medium sample spiked with 0.5 ppm are superimposed.

[0025] Figure 5 This is a superimposed chromatogram of the 2ppm standard solution and the culture medium sample provided according to the present invention. Detailed Implementation

[0026] While the invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that demonstrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter.

[0027] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0028] Example 1: Instruments, Reagents and Samples

[0029] 1. Instruments and Equipment

[0030] This embodiment uses the following instrument system, but the present invention is not limited to this specific model. Those skilled in the art can select equivalent equipment according to the laboratory conditions.

[0031] Ion chromatography system

[0032] The ion chromatography system is a Thermo Scientific™ Dionex™ ICS-6000 HPIC™ system or an equivalent ion chromatograph.

[0033] The system is equipped with a dual-piston tandem high-pressure gradient pump (Dionex™ EGC 600 Eluent Generator Cartridge for online generation of high-purity KOH eluent, or using pre-configured eluent bottles); an autosampler (Dionex™ AS-AP Autosampler) with a 100 μL injection loop and accuracy better than 0.5% RSD; a column oven (Dionex™ TCC-600 Thermostatted Column Compartment) with temperature control accuracy ±0.1℃; a conductivity detector (Dionex™ CD 600 Conductivity Detector) with an anion suppressor (Dionex™ AERS 600 Anion Electrolytically Regenerated Suppressor, 4 mm suppressor channel); and a chromatographic data processing system: Chromeleon™ 7.2.10 Chromatography Data System or equivalent software.

[0034] Sample pretreatment device

[0035] The RP pretreatment column used in the sample pretreatment device is a Waters Oasis HLB solid-phase extraction column (3 cc / 60 mg, Part No. WAT094225). Culture medium samples are pretreated with the RP pretreatment column before being fed into the chromatography system. The main function of the RP pretreatment column is to remove hydrophobic contaminants. The ion exchange column uses high-purity materials and utilizes the principles of reverse adsorption and ion exchange to remove interfering ions from various matrices, including anions, cations, metal ions, and hydrophobic impurities, thus avoiding contamination of the ion chromatography column and its impact on separation. The method of use is simple and easy to operate; liquid samples or extracts can be directly pushed through the column under pressure to achieve purification. Before use, the RP column needs to be activated. First, rinse the RP column with 5 mL of methanol, then rinse with 15 mL of high-purity water (>17 MΩ) at a flow rate of less than 3 mL / min. After rinsing, wait for 10 min, then take more than 3 mL of sample solution. No dilution is required. Pass the stock solution through the RP column. Do not collect the first 3 mL of sample. Collect the sample solution after 3 mL as the loading solution.

[0036] Auxiliary equipment

[0037] The analytical balance was a Mettler Toledo XP6 microbalance with an accuracy of 0.001 mg. The ultrapure water system was a Milli-Q Integral water purification system with a product water resistivity ≥18.2 MΩ·cm (25°C).

[0038] 2. Reagents and Standards

[0039] All experimental water was ultrapure water (resistivity ≥18.2 MΩ·cm) prepared using the above-described ultrapure water system. Potassium hydroxide (KOH): Fisher Chemical™ high-purity eluent concentrate (50% w / w, for ion chromatography, PNEL090350), or generated online using Dionex™ EGC 600 KOH capsules. Methanol (CH3OH): Fisher Chemical™, HPLC Grade, ≥99.9%.

[0040] 3. Chromatographic column

[0041] Analytical column: PrinCen PAS-19 anion exchange column (4.0 mm × 250 mm, 7 μm, Guangzhou Pulinsheng Technology Co., Ltd.).

[0042]

[0043] Guard column: PrinCen PAS-19 guard column (4.0 mm × 50 mm), used in conjunction with the analytical column.

[0044] 4. Sample

[0045] (1) Source of standard reagents

[0046] Nitric acid: Northern Weiye Metrology Group Co., Ltd., 1000 μg / mL;

[0047] Nitrous acid: Northern Weiye Measurement Group Co., Ltd., 1000 μg / mL;

[0048] Acetic acid: Aladdin; sodium acetate, anhydrous; pharmacopoeia grade.

[0049] (2) Preparation method of standard solutions: Prepare standard curves of 0.1 ppm, 0.2 ppm, 0.5 ppm, 1 ppm and 2 ppm.

[0050] A certain amount of sodium acetate was weighed and fixed to prepare a 1000 ppm acetate ion solution;

[0051] A mixed standard solution of acetate, nitrate, and nitrite ions was prepared by diluting sodium acetate, nitric acid, and nitrite at 1000 ppm to 10 mL.

[0052] The standard curve solutions were prepared by diluting a 10 ppm mixed standard solution of acetate, nitrate, and nitrite to obtain 0.1 ppm, 0.2 ppm, 0.5 ppm, 1 ppm, and 2 ppm solutions, respectively.

[0053] (3) Culture medium solution

[0054] The basal culture medium used was R2A (0.5 g yeast extract; 0.5 g peptone; 0.5 g casein hydrolysate; 0.5 g glucose; 0.5 g soluble starch; 0.3 g dipotassium hydrogen phosphate; 0.024 g anhydrous magnesium sulfate; 0.3 g sodium pyruvate; 1000 mL purified water; pH 7.2 ± 0.2 (25°C)). Samples 9-1 and 9-2 were prepared by diluting the above basal culture medium 10-fold.

[0055] (4) Spiked solution for culture medium

[0056] Use a pipette to transfer 0.05 mL of the 100 ppm (acetic acid, nitric acid, nitrite) mixed standard into a 10 mL centrifuge tube, then transfer 9.95 mL of the 10-fold diluted basic culture medium sample and shake well.

[0057] Example 2: Chromatographic Analysis Conditions

[0058] The core chromatographic analysis conditions of this invention are as follows, and all parameters have been systematically optimized, which is the key to achieving efficient separation.

[0059] 1. Eluent and gradient program

[0060] The eluent is generated online or pre-prepared using high-purity KOH and ultrapure water. The gradient program is carefully designed to achieve efficient separation of the three target analytes from the complex matrix. Figure 1 This is a chromatogram showing the detection of acetic acid, nitrite, and nitrate ions in a standard sample using the eluent conditions shown in the table below in Example 1. Figure 1 As shown, the acetic acid peak (16.255 min) and the nitrite peak (23.453 min) are well separated, and the nitrate peak (26.923 min) has a complete peak shape. All peaks are completely baseline separated and are not affected by other ions.

[0061]

[0062] 2. Instrument Configuration and Parameters

[0063] The injection volume was 25.0 μL (all-loop injection mode to avoid injection discrimination). The column temperature was 32.00 °C (precise temperature control is crucial for retention time reproducibility). The detector was a conductivity detector. The suppressor was a Dionex AERS 600 (4 mm) with a suppression current of 160 mA (this current was optimized to achieve the best balance between background conductivity, noise, and suppression capability). The cell temperature was 35.000 °C (slightly higher than the column temperature to effectively prevent bubble formation in the conductivity cell and ensure baseline stability). The data acquisition rate was 5 Hz. Under these conditions, the initial system back pressure was approximately ~2200 psi, which increased slightly during operation with increasing eluent concentration, within the normal range.

[0064] Example 3: System Suitability Testing and Method Validation

[0065] To demonstrate the reliability of this method, a series of mixed standard working solutions of varying concentrations were prepared and analyzed under the chromatographic conditions described above. Linear regression was performed using peak area (Y) against concentration (X, mg / L).

[0066]

[0067] The results showed that the three target compounds exhibited excellent linearity within their respective ranges, fully meeting the detection requirements from trace amounts to high concentrations in culture media.

[0068] To demonstrate the superiority of the technical solution of this invention, experimental data under other detection conditions were compared, with all other conditions (chromatographic column, column temperature, flow rate, and pretreatment method) remaining consistent.

[0069] Comparative Example 1

[0070] Gradients: 0-10 min (3 mM), 10.1-20 min (20 mM), 20.1-26 min (45 mM), 26.1-31 min (3 mM). Results are as follows: Figure 2 As shown, the acetic acid peak (16.076 min) and the nitrite peak (17.486 min) showed extremely poor separation (Rs < 0.8), almost overlapping each other; moreover, a large number of unseparated matrix interference peaks existed within this time period, making it impossible to accurately quantify either one. Although the nitrate peak (25.55 min) was able to elute, its peak shape broadened, and column efficiency decreased.

[0071] Comparative Example 2

[0072] Gradients: 0-4.5 min (10 mM), 4.5-14 min (20 mM), 14.1-24 min (45 mM), 24.1-29 min (10 mM). Results are as follows: Figure 3As shown, acetic acid eluted too early (6.681 min), co-eluting with numerous highly polar impurity ions (such as chloride ions and short-chain fatty acids) near the dead volume, making separation impossible. The separation of nitrous acid (11.952 min) and nitric acid (15.533 min) was acceptable, but the method failed because acetic acid could not be accurately quantified.

[0073] In comparison, the detection conditions listed in the embodiments of the present invention are the best scheme obtained through a large number of optimizations and screenings. Its time setting and concentration ramp-up rate are perfectly matched with the retention characteristics of the PAS-19 column, and it is the only and unpredictable successful combination to achieve the co-separation of three target substances and complex matrix.

[0074] Example 4: Sample Pretreatment Steps

[0075] Complex culture medium samples must undergo pretreatment to remove interfering substances such as proteins and hydrophobic macromolecules, protect the chromatographic column, and ensure quantitative accuracy. This invention employs the following standardized operating procedure:

[0076] Activation step: An Oasis HLB column (3cc / 60mg) was fixed onto a solid-phase extraction apparatus. 5.0 mL of methanol was slowly passed through the column at a flow rate of approximately 2 mL / min to solubilize and activate the hydrophobic groups on the stationary phase.

[0077] Equilibration step: Immediately afterwards, rinse the column with 15.0 mL of ultrapure water at a flow rate of approximately 3 mL / min to remove methanol and equilibrate the column environment to the aqueous phase in preparation for sample loading. After rinsing, keep the column moist and let it stand for 10 minutes to ensure that the stationary phase is fully hydrated.

[0078] Sample loading and washing procedures: Take an appropriate amount of cell culture medium sample, without dilution, and initially filter it through a 0.22 μm nylon needle filter to remove any possible small particles. Load 4.0-5.0 mL of the filtered sample onto the HLB column to ensure that the column packing reaches its saturation adsorption capacity while avoiding the loss of target analytes through penetration. Slowly pass the sample through the activated and equilibrated HLB column at a flow rate of 1-2 mL / min.

[0079] Collection procedure: Discard the first 3.0 mL of filtrate (this portion may contain unretained hydrophilic impurities and the flow rate is unstable). Collect the remaining eluent (usually another 2.0-3.0 mL) in a clean injection vial; this is the final test solution.

[0080] Column Cleaning and Storage: This column is for single use only and should be discarded after processing a single sample. If reuse is required, it must be thoroughly cleaned with 5 mL of methanol and 5 mL of methanol / water (50:50) solution, and cross-contamination should be assessed. However, for optimal reproducibility, single-use is recommended in this invention.

[0081] Oasis HLB packing material is a hydrophilic-lipophilic balanced polymer adsorbent. Through reflection mechanism and polar interaction, it can effectively capture proteins, hydrophobic pigments, lipids, and some additives in the culture medium. The target analytes, acetate, nitrite, and nitrate, are strongly hydrophilic ions and are not retained under aqueous conditions, directly penetrating the column to achieve purification. A 10-minute settling period is crucial to ensure sufficient contact between the packing material and the sample, reaching maximum adsorption capacity.

[0082] Example 5: Spiked Recovery Chromatogram (Optimized Conditions)

[0083] This embodiment aims to visually demonstrate, by comparing the chromatograms of the actual culture medium sample with those of the spiked sample, that the method of the present invention (using optimized conditions) can effectively eliminate matrix interference and ensure that there is no co-elution near the peaks of the target analytes (acetic acid, nitrite, nitric acid), thereby confirming the accuracy and specificity of quantification.

[0084] 1. Sample preparation

[0085] Take a cell culture medium sample and process it according to the sample pretreatment steps in Example 4 (purified using an Oasis HLB column). The resulting filtrate is used as the "unspecified sample". Take another cell culture medium sample from the same source and add standard solutions of acetate, nitrite, and nitrate to it. Then, perform the same HLB column pretreatment as described above on this spiked sample. The resulting filtrate is used as the "specified sample".

[0086] 2. Chromatographic conditions

[0087] The optimal chromatographic conditions of the present invention are fully followed as described in the foregoing embodiments:

[0088] Column: PrinCen PAS-19 (4.0×250mm)

[0089] Eluent gradient: as shown in Table 1

[0090] Flow rate: 1.000 mL / min

[0091] Column temperature: 32.00 °C

[0092] Suppressor current: 160 mA

[0093] Injection volume: 25.0 μL

[0094] 3. Operation and Atlas Acquisition

[0095] Unspecified and spiked samples were injected sequentially into the ion chromatography system, and their chromatograms were acquired separately. Using the overlay function of the chromatography workstation, the two chromatograms were aligned and superimposed on the same coordinate axis to obtain the desired result. Figure 4 .

[0096] 4. Results and Analysis

[0097] like Figure 4 As shown, the baselines of both chromatograms are stable with very few background impurity peaks. The peak shapes of the three target peaks in the spiked sample are completely consistent with the peak shapes at the corresponding positions in the unspiked sample, and the retention times show no drift (acetic acid ~16.3 min (visible after magnification), nitrite ~23.5 min, nitric acid ~26.9 min).

[0098] It can be clearly seen that at the target analyte peak position in the unspiked sample, the peak height and peak area of ​​the spiked sample show a significant and precise increase, while no new chromatographic peaks appear before or after this position, and the baselines completely overlap. There are no co-eluting matrix interference peaks near the target peak. If there were interfering substances, their response in the spiked sample chromatogram would not change, leading to shoulder peaks or peak shape distortion in the overlay plot, but this phenomenon is not observed in this chromatogram.

[0099] This method exhibits high specificity for the detection of acetic acid, nitrous acid, and nitric acid, with no interference from other components in the culture medium. HLB column pretreatment effectively removes matrix components that could interfere with detection, without adsorption loss of the analyte. The increase in peak area corresponds to a spiking dose of 0.5 mg / L, ensuring the accuracy of the quantitative results.

[0100] Example 6: Spiked Recovery Chromatogram (Unspiked Experiment)

[0101] This example aims to visually demonstrate, by comparing the chromatograms of standard solutions and culture medium samples under non-optimal gradient conditions (Comparative Example 2), that an unreasonable gradient program can lead to serious co-elution problems.

[0102] 1. Samples and Conditions

[0103] The standard solution was a mixed standard working solution of acetate, nitrite, and nitrate at a concentration of 2.0 mg / L. The culture medium sample was the "unspecified sample" after pretreatment as in Example 6.

[0104] 2. Chromatographic conditions

[0105] The elution procedure in Comparative Example 2 was used, and other parameters (chromatographic column, column temperature, flow rate, etc.) were kept consistent with the conditions of this invention.

[0106] Gradient procedure:

[0107] 0-4.5 min, 10 mM KOH;

[0108] 4.5-14 min, 20 mM KOH;

[0109] 14.1-24 min, 45 mM KOH;

[0110] 24.1-29 min, 10 mM KOH.

[0111] 3. Operation and Atlas Acquisition

[0112] The "2 mg / L standard solution" and "culture medium sample" were injected and analyzed separately under the conditions of Comparative Example 2, and chromatograms were collected. The two chromatograms were then overlaid to obtain the final result. Figure 5 .

[0113] 4. Results and Analysis

[0114] like Figure 5 As shown: At a low concentration of 2 mg / L, the acetate ion in the standard solution emitted a single peak at ~6.7 min, while nitrite and nitrate ions emitted peaks at ~12.0 min and ~15.5 min, respectively. Based solely on the standard solution, the separation appears acceptable. However, at the same position as the acetate standard peak (around ~6.7 min), the chromatogram of the culture medium sample shows a distinct peak with a shape completely different from the acetate peak in the standard solution.

[0115] The broad peak shape at 6.7 min in the culture medium sample indicates that acetate ions and one or more unknown matrix interference ions were eluted simultaneously at this point, forming an overlapping peak. This makes qualitative analysis by retention time impossible, and quantitative analysis by accurate integration is also impossible. The comparative conditions used a high initial concentration, causing weakly retained ions such as acetate to be eluted prematurely. Their retention time window overlaps with the elution times of numerous strongly polar matrix impurities (such as chloride ions, short-chain organic acids, etc.) near the dead volume, thus preventing separation. This figure is consistent with... Figure 4 This stark contrast demonstrates that the gradient strategy in this invention is a key and indispensable design element for overcoming matrix interference and achieving the specific separation of acetic acid. Those skilled in the art would hardly have anticipated through conventional reasoning that this particular gradient setup could solve the co-elution problem in this specific application. Figure 4 and Figure 5The direct evidence from both positive and negative perspectives confirms that the chromatographic method (including pretreatment, chromatographic column, and specific gradient program) provided by this invention has outstanding advantages in solving the analysis of target ions in complex matrix culture media, such as good separation, strong anti-interference ability, and accurate quantification. Its effect is far superior to other possible gradient conditions.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An ion chromatographic method for the simultaneous determination of acetate, nitrite and nitrate ions in complex matrix media, characterized in that, It comprises the following steps: (1) sample pretreatment: the culture medium sample is treated by a hydrophilic-lipophilic balance reversed-phase solid-phase extraction column, and the filtrate is collected as the test solution for the instrument; (2) chromatographic separation: the test solution obtained in step (1) is injected into an ion chromatography system, a high-capacity hydrophilic anion exchange chromatographic column is used, and potassium hydroxide solution is used as the eluent for gradient elution; (3) detection and analysis: the separated ions are detected by a conductivity detector, and quantitative analysis is performed by a standard curve method.

2. The ion chromatography method according to claim 1, characterized in that, The packing material of the high-capacity hydrophilic anion exchange chromatographic column is a polystyrene-divinylbenzene matrix with a crosslinking degree of 55%, and the particle size is 7 μm, and the theoretical plate number is not less than 7000.

3. The ion chromatography method of claim 1, wherein, The anion exchange chromatographic column is a PrinCen PAS-19 chromatographic column or an equivalent chromatographic column.

4. The ion chromatography method of claim 1, wherein, The hydrophilic-lipophilic balance reversed-phase solid-phase extraction column is an Oasis HLB column or an equivalent column; the pretreatment step comprises: first activating the column with methanol, then washing and balancing with ultrapure water, finally loading the culture medium sample, discarding the front part of the filtrate, and collecting the subsequent filtrate.

5. The ion chromatography method of claim 4, wherein, The amount of methanol used for activation is 3-5 mL, the amount of ultrapure water used for balancing is 10-15 mL, and the volume of the front part of the filtrate discarded is 2-3 mL.

6. The ion chromatographic method according to claim 1, characterized in that, The potassium hydroxide eluent is generated online or prepared in advance from high-purity potassium hydroxide concentrate.

7. The ion chromatography method of claim 1, wherein, The operating parameters of the ion chromatography system include: flow rate: 0.9-1.1 mL / min; column temperature: 30-35 ℃; injection volume: 20-30 μL; suppressor current: 150-170 mA; detector cell temperature: 33-37 ℃.

8. The ion chromatography method of claim 7, wherein, The operating parameters are: flow rate: 1.000 mL / min; column temperature: 32.00 ℃; injection volume: 25 μL; suppressor current: 160 mA; detector cell temperature: 35.000 ℃.

9. The ion chromatographic method according to any one of claims 1 to 8, characterized in that, The test solution obtained in step (1) is injected into an ion chromatography system, a PrinCen PAS-19 or an equivalent high-capacity hydrophilic anion exchange chromatographic column is used, potassium hydroxide solution is used as the eluent, and the following gradient program is used for elution: 0.0-15.0 min, the eluent concentration is 3 mmol / L; 15.1-25.0 min, the eluent concentration is linearly increased from 3 mmol / L to 20 mmol / L; 25.1-30.0 min, the eluent concentration is linearly increased from 20 mmol / L to 45 mmol / L; 30.1-35.0 min, the eluent concentration is reduced to 3 mmol / L and kept balanced.

10. The ion chromatography method according to any one of claims 1-8 is used for the quality control of biopharmaceutical, cell culture or microbial fermentation processes, for simultaneously monitoring the concentrations of acetate, nitrite and nitrate ions in the culture medium.

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