Method for detecting glucose in d-ribose fermentation liquor

Abstract

The invention relates to the technical field of glucose detection, and discloses a method for detecting glucose in d-ribose fermentation broth, which comprises the following steps: step 1, diluting a d-ribose fermentation broth sample and adjusting the d-ribose fermentation broth sample to a predetermined pH value to form a pretreated sample; 2, removing impurities and concentrating target components through solid-phase extraction to obtain a treated sample; 3, separating the sample by using high performance liquid chromatography, setting a mobile phase, a flow rate and a chromatographic column temperature, and separating to obtain a glucose component; and 4, injecting the separated components into a mass spectrum system on line, and detecting a characteristic ion signal of glucose. The high-sensitivity and accurate detection of glucose in the d-ribose fermentation liquor can be realized by adopting a high-performance liquid chromatography and mass spectrometry combined technology. Through optimized sample pretreatment and mobile phase selection, interference of other carbohydrates on glucose detection is reduced, and accurate determination of glucose concentration is ensured.

Description

Technical Field

[0001] The invention relates to the technical field of glucose detection, in particular to a method for detecting glucose in D-ribose fermentation broth. Background Art

[0002] With the development of biotechnology, fermentation broth is increasingly used in the pharmaceutical, food, environmental protection and other industries. In these industries, glucose, as one of the important raw materials, is often involved in the fermentation process, so the quantitative analysis of glucose is particularly important. The concentration of glucose directly affects the efficiency of the fermentation reaction and the quality of the final product. Therefore, the development of an efficient, sensitive and accurate glucose detection method is of great significance for optimizing the fermentation process, improving the yield and ensuring the product quality.

[0003] At present, the detection methods of glucose mainly include enzymatic method, electrochemical method, chromatography, etc. Among them, high performance liquid chromatography (HPLC) has been widely used in the detection of glucose due to its high resolution and high sensitivity. HPLC can accurately separate the glucose in the sample by using different types of chromatographic columns and mobile phases, and perform quantitative analysis in combination with ultraviolet detectors (UV) or mass spectrometry (MS). In particular, HPLC-MS technology combined with mass spectrometry has become the preferred method for glucose analysis in complex fermentation broths due to its highly sensitive and highly selective characteristics.

[0004] Although the existing HPLC-MS technology has high sensitivity and accuracy, there are still certain problems. Among the existing methods, most rely on complex sample pretreatment, such as solid phase extraction and buffer preparation, which not only increases the operational complexity of the experiment, but also may cause glucose loss in the sample. In addition, the existing chromatographic separation conditions have not been fully optimized, and there are certain errors in the separation effect and quantitative results of glucose in complex fermentation broth samples. Especially when the sample contains a variety of sugar substances, the peak shapes of glucose and other components may overlap, affecting the accuracy of the final detection. Therefore, there is an urgent need for a glucose detection method that can simplify the operating process, improve the separation effect and enhance the sensitivity, so as to improve the accuracy and efficiency of glucose detection in the fermentation broth. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for detecting glucose in D-ribose fermentation broth, which solves the problems of complex sample pretreatment, poor separation effect and insufficient sensitivity in the existing glucose detection method.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: A method for detecting glucose in D-ribose fermentation broth, comprising the following steps:

[0007] Step 1, diluting the d-ribose fermentation broth sample and adjusting it to a predetermined pH value to form a pretreated sample;

[0008] Step 2: removing impurities and concentrating target components by solid phase extraction to obtain a processed sample;

[0009] Step 3: Separate the sample using high performance liquid chromatography, set the mobile phase, flow rate and column temperature, and separate the glucose components;

[0010] Step 4: Inject the separated components into a mass spectrometer system online to detect characteristic ion signals of glucose;

[0011] Step 5: Calculate the glucose content in the sample by calibrating the sample signal and the standard curve.

[0012] Preferably, in step 1, the dilution volume ratio is 1:10, and the pH value of the sample is adjusted to 6.8±0.2 by adding 0.1% phosphoric acid.

[0013] Preferably, in step 2, the solid phase extraction uses a C18 solid phase extraction column, which specifically includes the following sub-steps:

[0014] The solid phase extraction column was activated with methanol;

[0015] The diluted sample was loaded and impurities were eluted with 10% ethanol;

[0016] The target component was eluted with 20% acetonitrile, concentrated and filtered.

[0017] Preferably, in step 3, the separation conditions of the high performance liquid chromatography include:

[0018] The chromatographic column is an amino column specially designed for sugars;

[0019] The mobile phase was acetonitrile-water (volume ratio 80:20), and 0.1% ammonia was added to adjust the pH to 7.5 ± 0.1;

[0020] The flow rate was 0.8 mL / min and the column temperature was 35 °C.

[0021] Preferably, in step 4, the ion source of the mass spectrometry system is an electrospray ionization source, and the multiple reaction monitoring mode is used to detect the characteristic ion signal of glucose, and the characteristic ion is [M+Na]^+=203.06m / z.

[0022] Preferably, in step 5, the glucose concentration is calculated by internal standard correction, and the correction formula is:

[0023]

[0024] Among them, R sampleis the target response signal in the sample, R blank is the blank sample signal, R internal is the internal standard signal, C internal is the known concentration of the internal standard.

[0025] Preferably, the standard curve is established using a series of glucose solutions with known concentrations in the range of 0.01-10 mg / mL, and the fitting formula is:

[0026] A=k·C+b

[0027] Where A is the peak area, C is the glucose concentration, k is the response coefficient, and b is the intercept.

[0028] Preferably, in the multiple reaction monitoring mode, samples are taken every 10 minutes by an automatic sampler, and a glucose concentration change curve is constructed, and the fitting formula is:

[0029] C(t)=C0·e -kt

[0030] Where C0 is the initial concentration, k is the degradation rate constant, and t is the time.

[0031] Preferably, the operating conditions of the mass spectrometry system include: a spray gas pressure of 40 psi, a drying gas flow rate of 12 L / min, a drying temperature of 350°C, and a mass spectrometry scanning range of m / z 150-300.

[0032] Preferably, the detection limit is 0.01 mg / mL, the quantification limit is 0.1 mg / mL, and the recovery rate is 98.5%±1.2%.

[0033] The present invention provides a method for detecting glucose in D-ribose fermentation broth. The method has the following beneficial effects:

[0034] 1. The present invention can achieve high sensitivity and accurate detection of glucose in d-ribose fermentation broth by using high performance liquid chromatography-mass spectrometry. By optimizing sample pretreatment and mobile phase selection, the interference of other sugar substances on glucose detection is reduced, ensuring the accurate determination of glucose concentration. This enables the method to maintain high sensitivity in low-concentration glucose samples, meeting the high requirements of experimental and industrial applications.

[0035] 2. The present invention simplifies the sample pretreatment steps and removes the complex extraction and purification steps in the traditional method, making the detection process simpler and more efficient. By directly diluting and adjusting the pH of the fermentation broth sample and combining it with high-performance liquid chromatography separation technology, it not only reduces reagents and time consumption, but also reduces the technical requirements of operators, which is particularly suitable for high-throughput detection applications.

[0036] 3. The present invention uses optimized mobile phase and chromatographic column selection in high performance liquid chromatography separation, effectively separates glucose from other components in the fermentation broth, and reduces interference caused by sample complexity. Even in highly complex samples, high separation and good peak shape can still be maintained, ensuring the reliability of the analysis results. This technology can be widely used in different types of fermentation broth analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Embodiment 1:

[0040] Please see attached Figure 1 The present invention provides a method for detecting glucose in a D-ribose fermentation broth, comprising the following steps:

[0041] Step 1: Sample pretreatment

[0042] Regarding step 1: sample pretreatment, the present invention first needs to dilute, adjust pH and remove impurities from the d-ribose fermentation broth sample as necessary. The purpose of these pretreatment steps is to remove impurities and interfering components in the fermentation broth so that the sample is suitable for subsequent high performance liquid chromatography (HPLC) analysis and mass spectrometry detection. By pretreating the sample, the accuracy and sensitivity of the detection can be improved, while ensuring the reliability of the subsequent analysis results.

[0043] 1. Sample dilution and pH adjustment

[0044] Before analyzing the d-ribose fermentation broth sample, the sample is first diluted to reduce the concentration of interfering substances that may exist in the sample and make it meet the needs of subsequent analysis. The specific operation is: dilute the d-ribose fermentation broth sample with deionized water at a volume ratio of 1:10, the purpose is to reduce the concentration of the sample by dilution, and reduce the interference of high concentrations of salt and inorganic substances that may exist in the sample on subsequent analysis.

[0045] Subsequently, the pH value of the diluted sample is adjusted. According to the experimental requirements, the pH of the sample is adjusted to 6.8±0.2 by adding 0.1% phosphoric acid solution. This pH range helps the stability of glucose during liquid chromatography analysis, avoiding glucose decomposition or affecting the subsequent analysis process due to too low or too high pH. This step ensures that the structure and properties of glucose are not affected and can stably enter the separation and detection stage.

[0046] 2. Solid phase extraction to remove impurities

[0047] In order to remove impurities such as proteins, pigments, inorganic salts, etc. in the samples and reduce the influence of these interfering substances on glucose determination, solid phase extraction (SPE) technology was used to process the samples. The specific implementation steps are as follows:

[0048] Activation and equilibration: Use C18 solid phase extraction column (5μm, ) for sample treatment. First, the solid phase extraction column is activated with methanol to ensure that the filler in the column has good adsorption performance. Subsequently, the solid phase extraction column is equilibrated with deionized water to prepare for receiving the sample.

[0049] Loading sample: Load the diluted and pH-adjusted sample onto the SPE column. At this point, glucose and other water-soluble substances will be adsorbed by the C18 filler in the column, while non-target components (such as impurities, salts, etc.) will be eluted.

[0050] Elution of impurities: The column was eluted with 10% ethanol solution to remove low polar impurities and other interfering components in the sample. Subsequently, 20% acetonitrile solution was used for elution, which eluted glucose from the C18 column.

[0051] Concentration and filtration: The eluate is concentrated to about 1 mL by a vacuum concentrator, and the concentrated liquid is filtered using a 0.22 μm filter membrane to remove remaining solid particles and impurities. This step ensures that the glucose component in the sample enters the subsequent separation and analysis process and reduces possible interfering substances.

[0052] 3. Addition of internal standard substances

[0053] In order to improve the accuracy and reliability of quantification in subsequent mass spectrometry analysis, internal standard substances are added during the sample pretreatment stage. 13C6-glucose is often used as an internal standard, which has similar chemical properties to glucose, but different mass spectrometry signals from glucose. Therefore, the internal standard can be used to correct errors caused by instrument fluctuations, losses during analysis, etc. during mass spectrometry detection.

[0054] The amount of internal standard added is usually 1% of the glucose concentration. The concentration of glucose in the sample can be calculated more accurately by performing correction based on the signal intensity ratio of the internal standard and the glucose in the sample.

[0055] In order to improve the accuracy of the measurement, in this embodiment, the internal standard method is used for data correction. The ratio of the internal standard signal to the glucose signal in the sample is used to correct the error in the sample signal. The specific correction formula is as follows:

[0056]

[0057] Among them, R sample is the target response signal in the sample, R blank is the blank sample signal, R internal is the internal standard signal, C internal is the known concentration of the internal standard, C sample is the concentration of glucose in the sample to be tested.

[0058] This formula can achieve accurate quantification of the glucose concentration in the sample while reducing the result deviation caused by sample complexity or operational errors.

[0059] Through the sample pretreatment in step 1 above, the glucose in the d-ribose fermentation broth is effectively separated and purified. Through a series of operations such as dilution, pH adjustment, solid phase extraction to remove impurities, and internal standard addition, the purity of the sample and the accuracy of the detection are ensured, laying a good foundation for subsequent high-performance liquid chromatography and mass spectrometry analysis. These technical steps not only ensure the stability of glucose and the reliability of the measurement results, but also effectively eliminate possible interference factors, so that the final glucose concentration analysis results have high sensitivity and accuracy.

[0060] Step 2: HPLC separation

[0061] For step 2: high performance liquid chromatography separation, the present invention uses high performance liquid chromatography (HPLC) technology to separate the sample to achieve effective separation of glucose from other components. By optimizing the chromatographic conditions, it is possible to ensure good separation of glucose on the chromatographic column, reduce interference, obtain a high-purity glucose sample, and thereby improve the accuracy and sensitivity of mass spectrometry analysis.

[0062] 1. Selection of chromatographic columns and device configuration

[0063] In this embodiment, an amino column specially used for separation of sugars is used as a chromatographic column. The size of the chromatographic column is 250 mm × 4.6 mm, the filler particle size is 5 μm, and it has a high separation efficiency and is suitable for separating water-soluble sugar substances such as glucose. As a common sugar separation material, the amino column has good selectivity and can effectively separate the interference of glucose and other similar molecules.

[0064] The HPLC system includes a high-pressure pump, an automatic sampler, a detector, and a chromatographic column. The mobile phase is transported to the chromatographic column by the pump system, and the pre-treated sample is injected into the system through the sample injector in front of the column. When the sample passes through the chromatographic column, different components are separated in the chromatographic column according to the differences in properties such as molecular size and polarity. The separated components are detected by UV or mass spectrometry detectors.

[0065] 2. Selection and preparation of mobile phase

[0066] In order to achieve a good separation of glucose from other components, the selection of mobile phase is crucial. In this embodiment, the mobile phase consists of acetonitrile and water, and 0.1% ammonia water is added to adjust the pH value. The specific configuration is as follows:

[0067] The mobile phase is acetonitrile-water (volume ratio 80:20). As an organic solvent, acetonitrile can effectively dissolve non-polar substances in the sample and improve the efficiency of chromatographic separation.

[0068] 0.1% ammonia water is used to adjust the pH to 7.5±0.1. This pH value helps the stability of glucose in the chromatographic column and can reduce the interaction between other components and the chromatographic column, thereby optimizing the separation effect.

[0069] The composition and pH adjustment of the mobile phase ensure that the distribution coefficient of glucose in the chromatographic column is appropriate, thereby avoiding too fast elution or too slow separation, and ensuring that glucose can be clearly separated from other components to form clear chromatographic peaks.

[0070] 3. Optimization and setting of separation conditions

[0071] In the HPLC separation process, the separation efficiency is affected by many factors, including flow rate, column temperature and sample loading amount. In this embodiment, the optimized separation conditions are as follows:

[0072] Flow rate setting: The chromatographic flow rate is 0.8 mL / min. This flow rate can avoid too long sample analysis time or too high chromatographic column pressure while ensuring separation efficiency. This flow rate helps to generate enough separation time between glucose and other sugar substances, so that glucose can be better separated.

[0073] Column temperature setting: The column temperature is set to 35°C. At this temperature, the chromatographic separation effect is the best, which can ensure the stability of the filler in the column and the separation efficiency, and avoid the influence of excessive temperature on the sample or solvent. Under normal temperature conditions, the chromatographic column temperature is controlled at 35°C to effectively avoid the loss of column efficiency and the decrease of separation efficiency.

[0074] Sample injection volume: The sample injection volume is 20 μL. This injection volume can ensure that the glucose signal intensity is moderate and will not cause chromatographic peak tailing or unclear separation due to excessive injection.

[0075] By accurately setting these separation conditions, this embodiment can ensure the efficient separation of glucose, avoid the interference of other components on glucose, and make the chromatographic peak of each component clearly identifiable.

[0076] 4. Glucose Peak Monitoring During Separation

[0077] During the HPLC separation process, the separation time of glucose is about 4.5±0.2 minutes. The chromatographic peak signal obtained by online monitoring of the UV detector or mass spectrometry (MS) system can detect an obvious response signal when the characteristic peak of glucose appears. Due to the high resolution of the mobile phase conditions and chromatographic column selection, the chromatographic peak of glucose can be clearly separated and there is no obvious overlap with other components.

[0078] The chromatographic peak of glucose has a good shape, obvious symmetry and narrow peak width, indicating that the separation process is sufficient and the purity of glucose is high. To further ensure the accuracy of quantitative analysis, the internal standard substance 13C6-glucose is used for correction in this embodiment to compensate for signal deviations caused by factors such as injection amount error and instrument fluctuation.

[0079] 5. Data recording and processing

[0080] The separated glucose sample is further analyzed by a mass spectrometry detection system. In this step, the chromatographic peak area is compared with the signal of the internal standard, and the concentration of glucose is calculated using the internal standard correction formula mentioned above. By using the standard curve of the standard glucose solution, the accurate quantification of the glucose concentration in the sample can be achieved.

[0081] Through the analytical method of high performance liquid chromatography combined with mass spectrometry, efficient and accurate determination of glucose concentration can be achieved, further improving the sensitivity and reliability of detection.

[0082] Step 3: Mass spectrometry detection

[0083] For step 3: mass spectrometry detection, the present invention uses mass spectrometry (MS) technology to further analyze the glucose components separated by high performance liquid chromatography (HPLC). Mass spectrometry can provide higher sensitivity and accuracy, accurately determining the concentration of glucose in a qualitative and quantitative manner. Through the multiple reaction monitoring (MRM) mode of mass spectrometry, the characteristic ions of glucose can be accurately captured in a complex fermentation broth, thereby improving the accuracy of detection.

[0084] 1. Selection of mass spectrometer and ionization source

[0085] In order to improve the detection sensitivity of glucose, an electrospray ionization source (ESI) was used. ESI is an ionization technology widely used in liquid chromatography-mass spectrometry (LC-MS), which can generate high-intensity ion signals at low sample concentrations and is suitable for the analysis of small molecules such as sugars and peptides.

[0086] The working principle of the ES I source is to spray the liquid sample and charge it under the action of the electric field, and then enter the mass spectrometer for analysis. In this embodiment, the positive ion mode is used for analysis because glucose has a stronger response in the positive ion mode and can provide a more stable ion signal.

[0087] 2. Multiple Reaction Monitoring (MRM) Mode

[0088] The multiple reaction monitoring (MRM) mode of mass spectrometry was used to obtain higher sensitivity and selectivity. The MRM mode can simultaneously monitor the characteristic ions of glucose and internal standard 13C6-glucose and perform selective scanning in different mass-to-charge ratio (m / z) ranges.

[0089] The characteristic ion of glucose is [M+Na]^+=203.06m / z, while the characteristic ion of the internal standard 13C6-glucose is [M+Na]^+=209.06m / z. By monitoring these two ions, accurate quantitative analysis can be performed.

[0090] 3. Mass spectrometry analysis parameter setting

[0091] In mass spectrometry analysis, in order to ensure clear ion signals of glucose and internal standard substances, the parameters of the mass spectrometer need to be optimized according to the properties of the target. The specific parameter settings are as follows:

[0092] Spray gas pressure: 40 psi. This spray gas pressure helps to stably vaporize and ionize the sample and improve signal stability.

[0093] Drying gas flow rate: 12L / min. This gas flow rate ensures that the sample can be quickly vaporized during the spray process to avoid the formation of particles or unstable ion groups.

[0094] Drying temperature: 350°C. This temperature can effectively remove moisture from the solvent, ensure stable ionization of glucose molecules, and avoid glucose degradation or ion signal loss caused by excessive temperature.

[0095] 4. Data Collection and Processing

[0096] The mass spectrometry data were collected in scanning mode, with selective scanning in the m / z range of 150–300. The characteristic ions of glucose and the ions of the internal standard were monitored in MRM mode, which could effectively distinguish glucose from other possible interfering substances.

[0097] During data processing, the concentration of glucose is calculated by the peak area ratio. The specific calculation formula is as follows:

[0098]

[0099] in:

[0100] C sample is the concentration of glucose in the sample,

[0101] A sample is the peak area of ​​glucose in the sample,

[0102] C internal is the known concentration of the internal standard substance,

[0103] A internal is the peak area of ​​the internal standard substance.

[0104] Through the internal standard method of correction, factors such as instrument fluctuations and injection errors in the sample can be compensated, thereby improving the accuracy of quantitative analysis.

[0105] Step 4: Quantification and data analysis

[0106] Regarding step 4: data analysis and result reporting, the present invention further processes the raw data obtained by mass spectrometry detection and calculates the concentration of glucose. The data analysis includes drawing a standard curve, quantitative calculation of the glucose concentration, statistical processing of the analysis results, etc., and finally obtains the concentration of glucose in the fermentation broth.

[0107] 1. Drawing of standard curve

[0108] To ensure the accuracy of the quantitative results, it is first necessary to draw a standard curve using a glucose standard solution of known concentration. The concentration range of the standard solution should cover the possible glucose concentration of the sample to be tested, usually set between 0.1mM and 10mM. The peak area of ​​each standard solution must be obtained during HPLC and mass spectrometry detection.

[0109] The glucose concentration in unknown samples can be calculated using the standard curve.

[0110] 2. Calculation of Glucose Concentration

[0111] The actual concentration of glucose is calculated by inputting the peak area ratio data of glucose and internal standard substance in the sample through data processing software or Excel and other tools. Using the previously established standard curve, the peak area of ​​the sample is substituted into the formula to obtain the concentration of glucose in the sample.

[0112] 3. Results statistics and reports

[0113] In the analysis results, the glucose concentrations in different samples will be statistically analyzed, and the average value, standard deviation and other data will be given. These data can be used to monitor the fermentation process and determine whether the fermentation has reached the expected glucose consumption level or whether the fermentation conditions need to be further optimized.

[0114] In the report, all experimental conditions, relevant parameters of the standard curve, the use of internal standard substances, and the specific analysis steps of the samples should also be noted. By recording each step and data in detail, data support can be provided for subsequent experiments and the reproducibility of the results can be ensured.

[0115] Embodiment 2:

[0116] As in Example 1, this example also uses high performance liquid chromatography (HPLC) combined with mass spectrometry (MS) to detect glucose in d-ribose fermentation broth. However, in Example 2, the process is slightly simplified, and only one variable (e.g., mobile phase composition) is used for optimization. In this example, the separation effect of glucose is slightly inferior to that of Example 1, but still has certain applicability in terms of sensitivity and efficiency of detection.

[0117] Step 1: Sample pretreatment

[0118] As in Example 1, in this example, the d-ribose fermentation broth sample is first diluted, pH adjusted, and solid phase extracted to remove impurities in the sample. The sample pretreatment operation steps are the same as in Example 1, and the specific method is described in step 1, mainly using a diluent and a solid phase extraction column to remove impurities and interfering components.

[0119] 1. Sample dilution and pH adjustment:

[0120] Dilute the D-ribose fermentation broth at a ratio of 1:10.

[0121] Adjust the pH to 6.8 ± 0.2 using phosphoric acid solution.

[0122] 2. Solid phase extraction to remove impurities:

[0123] A C18 solid phase extraction column was used, and the activation and elution steps were the same as those in Example 1.

[0124] After the samples were concentrated and filtered, internal standard 13C6-glucose was added for calibration.

[0125] Step 2: HPLC separation

[0126] In this embodiment, the composition of the mobile phase is slightly simplified compared with the first embodiment. The mobile phase is acetonitrile-water (volume ratio 75:25), and ammonia water is not added to adjust the pH, but the distribution coefficient of the sample is controlled by the ratio of acetonitrile and water. Although the simplification of the mobile phase reduces the separation effect, glucose and other sugar substances can still be separated within a certain range.

[0127] 1. Selection of chromatographic column:

[0128] An amino column (250 mm×4.6 mm, 5 μm filler) was used for separation, and the column selection in Example 1 was maintained.

[0129] 2. Choice of mobile phase:

[0130] The mobile phase is composed of acetonitrile and water in a volume ratio of 75:25, and no ammonia is added to adjust the pH. This mobile phase composition simplifies the treatment process, but may result in a less effective separation of sugars than in Example 1, especially in the case of interference from high-concentration impurities, where the separation effect of glucose is poor.

[0131] 3. Separation conditions:

[0132] The flow rate was 1.0 mL / min, the column temperature was set at 30° C., and the injection volume was 30 μL. The separation conditions were simplified at a higher flow rate, which reduced the analysis time but may result in a lower separation degree than in Example 1.

[0133] Step 3: Mass spectrometry detection

[0134] Mass spectrometry detection in this embodiment is the same as in Example 1, and still uses an electrospray ionization source (ESI) and a multiple reaction monitoring (MRM) mode. The characteristic ions of glucose and internal standard 13C6-glucose are [M+Na]^+=203.06m / z and [M+Na]^+=209.06m / z, respectively. Data acquisition uses the MRM mode to ensure high sensitivity.

[0135] 1. Ionization source and detection mode:

[0136] The electrospray ionization source (ESI) adopts positive ion mode, and maintains the same working principle as in Example 1. The ion signals of glucose and internal standard are respectively monitored in MRM mode.

[0137] 2. Data processing:

[0138] The internal standard method was used for quantitative calculation, and the formula was the same as that in Example 1. Due to the change of the mobile phase, the chromatographic peak width of glucose may be slightly larger, resulting in a slightly lower signal accuracy than that in Example 1.

[0139] Step 4: Data analysis and result reporting

[0140] As in Example 1, quantitative calculation was performed using the glucose standard curve during data analysis. The standard curve was drawn using the same method as in Example 1, using a glucose standard solution and calibrated using an internal standard substance.

[0141] Since a simpler mobile phase is used in Example 2, the separation effect is slightly weakened, so the linearity and precision of the obtained standard curve may be lower than that of Example 1. However, this method can still accurately determine the glucose concentration in the sample, and the operation process is simplified, which is suitable for some application scenarios that do not require extremely high separation.

[0142] The formula for drawing the standard curve is:

[0143] A=k·C+b

[0144] Among them, A is the peak area, C is the glucose concentration, k and b are constants in the regression equation.

[0145] Table 1 Comparison table of experiments of embodiment 1 and embodiment 2

[0146]

[0147]

[0148] The regression coefficient of the standard curve (R 2 ): The regression coefficient of Example 1 is close to 1, indicating that the linearity of its standard curve is good; while the regression coefficient of Example 2 is slightly lower, indicating that the linearity of the standard curve is poor, which may lead to an increase in the error of quantitative analysis.

[0149] Separation effect and peak shape: The separation effect of Example 1 is significantly better than that of Example 2, and the symmetry and separation degree of the glucose peak are better. In Example 2, although glucose can be separated, the peak shape is slightly wide and there is a slight tailing, which affects the separation effect.

[0150] Sensitivity and detection limit: Example 1 has higher sensitivity in the detection of low-concentration glucose and can still obtain reliable detection results at lower concentrations; while Example 2 has lower sensitivity and may not be able to detect very low concentrations of glucose, and is suitable for more concentrated samples.

[0151] Due to the optimized mobile phase conditions, good separation effect and high sensitivity, Example 1 is suitable for accurate detection of glucose in complex samples, especially in low-concentration samples, and its effect is better than that of Example 2.

[0152] Although the second embodiment is simpler in operation and the analysis time is shorter, it is slightly inferior to the first embodiment in separation effect, sensitivity and precision. Therefore, it is more suitable for analysis of samples that do not require extremely high precision or are relatively simple.

[0153] Embodiment three:

[0154] In Example 3, the present invention still uses high performance liquid chromatography (HPLC) combined with mass spectrometry (MS) technology to detect glucose in d-ribose fermentation broth. Compared with Example 1, Example 3 simplifies the sample pretreatment and separation conditions, resulting in reduced separation effect and sensitivity. Example 3 is suitable for relatively simple sample analysis with less interference, but may not achieve the same effect as Example 1 in complex samples.

[0155] Step 1: Sample pretreatment

[0156] In this embodiment, in order to simplify the sample pretreatment process, no solid phase extraction step was performed, and the sample was directly diluted and pH adjusted. Although solid phase extraction was omitted, impurities in the fermentation broth may not be fully removed, thereby affecting the accuracy of subsequent analysis.

[0157] 1. Sample dilution and pH adjustment:

[0158] Dilute the D-ribose fermentation broth at a ratio of 1:5.

[0159] The pH was adjusted to 6.8 ± 0.2 using sodium hydroxide solution without using a buffer.

[0160] 2. Solid phase extraction omitted:

[0161] In order to simplify the operation, this embodiment directly performs pH adjustment and dilution without performing solid phase extraction treatment, which may result in that some interfering substances in the sample are not completely removed, affecting subsequent analysis.

[0162] Step 2: HPLC separation

[0163] Compared with Example 1, the selection of the chromatographic column and mobile phase used in this example is relatively simple. A common C18 chromatographic column is used, the mobile phase is composed of acetonitrile and water, and the mobile phase is not optimized, which may result in poor separation effect of glucose.

[0164] 1. Selection of chromatographic column:

[0165] An ordinary C18 chromatographic column (250 mm × 4.6 mm, 5 μm filler) was used for separation without using an amino column or other special columns, resulting in poor separation results.

[0166] 2. Choice of mobile phase:

[0167] The mobile phase consists of acetonitrile and water in a volume ratio of 70:30. Compared with the optimized mobile phase in Example 1, it has a stronger polarity, which may affect the distribution of glucose and other substances, resulting in a separation effect that is not as good as that in Example 1.

[0168] 3. Separation conditions:

[0169] The flow rate was 1.0 mL / min, the column temperature was set at 25°C, and the injection volume was 50 μL. Lower column temperature and injection volume may lead to poor separation effect, especially when glucose is close to other sugar substances, it is difficult to achieve complete separation.

[0170] Step 3: Mass spectrometry detection

[0171] In this example, the mass spectrometry analysis conditions were kept the same as those in Example 1. However, due to the poor separation effect of glucose, the mass spectrometry signal intensity was low, which affected the final quantitative result.

[0172] 1. Ionization source and detection mode:

[0173] Electrospray ionization (ESI) was used for positive ion analysis, and the mass spectrometry parameters were consistent with those in Example 1.

[0174] 2. Data collection and processing:

[0175] The data were collected in multiple reaction monitoring (MRM) mode, monitoring the characteristic ions of glucose and internal standard 13C6-glucose. However, due to the poor separation of glucose, the reliability and accuracy of the mass spectrometry data were low.

[0176] Step 4: Data analysis and result reporting

[0177] Similar to Example 1, the standard curve method was used for quantitative analysis. However, due to the incomplete separation of glucose, the analysis results of the actual samples had large errors and low accuracy.

[0178] 1. Standard curve drawing:

[0179] The method for drawing the standard curve is the same as that in Example 1, using a glucose standard solution of known concentration to draw the standard curve. However, due to the poor separation effect in Example 3, the accuracy of the standard curve is low, which may lead to errors in the quantitative results.

[0180] 2. Data processing:

[0181] The internal standard method is used for data correction. Due to poor separation, the interference of the internal standard may increase, resulting in deviations in the calculation results.

[0182] Table 2 Comparison table of experimental data of Examples 1 and 3

[0183]

[0184]

[0185] The regression coefficient of the standard curve (R2 ): The regression coefficient of Example 1 is close to 1, and the linearity of the standard curve is good, while the regression coefficient of Example 3 is significantly lower than that of Example 1, indicating that its quantitative analysis accuracy is poor.

[0186] Separation effect and peak shape: The separation effect of Example 1 is good, the glucose peak is clear and symmetrical, and completely separated. However, the separation effect of Example 3 is poor, the glucose peak is wide and has obvious tailing phenomenon, and has a large overlap with other substances, which affects the accuracy of subsequent analysis.

[0187] Sensitivity and detection limit: Example 1 can detect glucose at a relatively low concentration, while Example 3 has a lower sensitivity and a relatively high detection limit and cannot achieve the same sensitivity in complex samples.

[0188] Analysis time: The analysis time of Example 3 was slightly longer, mainly due to the poor separation effect and the longer peak width.

[0189] Embodiment 1 Due to the optimized separation conditions and sensitivity, it can provide relatively accurate quantitative results and is suitable for complex sample analysis with higher requirements.

[0190] Although Example 3 simplifies the sample pretreatment and separation steps, it is only suitable for simple sample analysis due to its poor separation effect and low sensitivity, and is not suitable for accurate detection of glucose in complex samples.

[0191] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting glucose in a D-ribose fermentation broth, characterized in that: The following steps are involved: Step 1, diluting the d-ribose fermentation broth sample and adjusting it to a predetermined pH value to form a pretreated sample; Step 2: removing impurities and concentrating target components by solid phase extraction to obtain a processed sample; Step 3: Separate the sample using high performance liquid chromatography, set the mobile phase, flow rate and column temperature, and separate the glucose components; Step 4: Inject the separated components into a mass spectrometer system online to detect characteristic ion signals of glucose; Step 5: Calculate the glucose content in the sample by calibrating the sample signal and the standard curve.

2. The method for detecting glucose in a D-ribose fermentation broth according to claim 1, characterized in that: In step 1, the dilution volume ratio is 1:10, and the pH value of the sample is adjusted to 6.8±0.2 by adding 0.1% phosphoric acid.

3. The method for detecting glucose in a D-ribose fermentation broth according to claim 1, characterized in that: In step 2, the solid phase extraction uses a C18 solid phase extraction column, which specifically includes the following sub-steps: The solid phase extraction column was activated with methanol; The diluted sample was loaded and impurities were eluted with 10% ethanol; The target component was eluted with 20% acetonitrile, concentrated and filtered.

4. The method for detecting glucose in a D-ribose fermentation broth according to claim 1, characterized in that: In step 3, the separation conditions of the high performance liquid chromatography include: The chromatographic column is an amino column specially designed for sugars; The mobile phase was acetonitrile-water (volume ratio 80:20), and 0.1% ammonia was added to adjust the pH to 7.5 ± 0.1; The flow rate was 0.8 mL / min and the column temperature was 35 °C.

5. The method for detecting glucose in a D-ribose fermentation broth according to claim 1, characterized in that: In step 4, the ion source of the mass spectrometry system is an electrospray ionization source, and the multiple reaction monitoring mode is used to detect the characteristic ion signal of glucose, and the characteristic ion is [M+Na]^+=203.06m / z.

6. The method for detecting glucose in a D-ribose fermentation broth according to claim 1, characterized in that: In step 5, the glucose concentration is calculated by internal standard correction, and the correction formula is: Among them, R sample is the target response signal in the sample, R blank is the blank sample signal, R internal is the internal standard signal, C internal is the known concentration of the internal standard.

7. The method for detecting glucose in a D-ribose fermentation broth according to claim 1, characterized in that: The standard curve is established using a series of glucose solutions with known concentrations ranging from 0.01 to 10 mg / mL, and the fitting formula is: A=k·C+b Where A is the peak area, C is the glucose concentration, k is the response coefficient, and b is the intercept.

8. The method for detecting glucose in D-ribose fermentation broth according to claim 1, characterized in that: In the multiple reaction monitoring mode, samples were taken every 10 minutes by an automatic sampler, and a glucose concentration curve was constructed. The fitting formula was: C(t)=C0·e -kt Where C0 is the initial concentration, k is the degradation rate constant, and t is the time.

9. The method for detecting glucose in D-ribose fermentation broth according to claim 1, characterized in that: The operating conditions of the mass spectrometry system included: spray pressure of 40 psi, drying gas flow rate of 12 L / min, drying temperature of 350 °C, and mass spectrometry scanning range of m / z 150–300.

10. The method for detecting glucose in D-ribose fermentation broth according to claim 1, characterized in that: The detection limit was 0.01 mg / mL, the quantification limit was 0.1 mg / mL, and the recovery rate was 98.5%±1.2%.

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