Amino acid detection method of online pre-column derivatization coupled with liquid chromatography-mass spectrometry

Abstract

This invention discloses an online pre-column derivatization coupled with liquid chromatography-mass spectrometry (LC-MS) for amino acid detection, relating to the field of analytical detection technology. The method includes: obtaining a sample for protein hydrolysis to obtain a hydrolysate; mixing the hydrolysate with a derivatizing reagent and performing an online derivatization reaction through multiple reaction zones arranged in series, each zone maintaining a different pH environment; setting an online detection unit at the reaction zone outlet to monitor the concentration of the derivatized product and adjust the reaction conditions in real time; performing chromatographic separation of the derivatized reaction solution; switching the ionization polarity mode of the ion source according to the elution time window for mass spectrometry detection; and substituting the mass spectrometry response signal into a standard curve to calculate the amino acid concentration value. This invention achieves automated derivatization and high-sensitivity detection of amino acids, with advantages such as simple operation, controllable reaction conditions, and high detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analytical detection, in particular, to an amino acid detection method of online pre-column derivatization coupled with liquid mass spectrometry. BACKGROUND

[0002] At present, the amino acid detection methods mainly include ion exchange chromatography, gas chromatography and liquid chromatography. The liquid chromatography is widely applied to the amino acid detection because of its good separation effect and simple operation. The traditional amino acid liquid chromatography detection needs to carry out pre-column derivatization, and the commonly used derivatization reagents include o-phthaldehyde, dinitrofluorobenzene and the like. These methods have problems of difficult control of derivatization reaction conditions, poor stability of derivatization products and low sensitivity.

[0003] The current liquid mass spectrometry detection method still needs manual sample pretreatment and derivatization reaction, and the operation is complicated, and the derivatization reaction conditions are difficult to accurately control, so that the reproducibility of the detection result is poor. Because the dissociation constants of different amino acids are quite different, it is difficult to ensure that all amino acids can obtain good derivatization efficiency by using a single pH condition for derivatization reaction.

[0004] The existing detection method usually adopts a fixed mass spectrometry detection mode, and cannot be optimized according to the ionization characteristics of different amino acid derivatives, which affects the detection sensitivity. The existing technology also has problems of incomplete derivatization reaction, low detection efficiency and the like, and it is difficult to meet the demand of rapid and accurate detection. SUMMARY

[0005] The purpose of the present application is to provide an amino acid detection method of online pre-column derivatization coupled with liquid mass spectrometry, which aims to at least solve one of the technical problems existing in the prior art.

[0006] The technical scheme of the present application is as follows: an amino acid detection method of online pre-column derivatization coupled with liquid mass spectrometry, comprising the following steps:

[0007] Obtaining a sample to be detected and carrying out protein hydrolysis treatment to obtain a hydrolysate containing free amino acids;

[0008] Mixing the hydrolysate with a derivatization reagent, and then sequentially passing through a plurality of reaction zones arranged in series to carry out online derivatization reaction, each reaction zone maintaining a different pH environment for different dissociation constant amino acids, to obtain a derivatization reaction liquid containing amino acid derivatives;

[0009] An online detection unit is arranged at the outlet of the plurality of reaction zones to monitor the generation concentration of the amino acid derivative products, and the derivatization reagent supply rate and the reaction zone temperature are adjusted in real time according to the generation concentration, to obtain a mobile phase containing amino acid derivatives;

[0010] Carrying out chromatographic separation on the mobile phase containing amino acid derivatives to obtain amino acid derivative components separated in the order of elution time;

[0011] Based on the elution time window of the amino acid derivative components, the ionization polarity mode of the ion source is switched during the mass spectrometry detection process so that the amino acid derivative components are detected in the mode with the optimal ionization efficiency, and the mass spectrometry response signal is obtained.

[0012] Substitute the mass spectrometry response signal into the preset standard curve to calculate the amino acid concentration value corresponding to the amino acid derivative component in the sample to be tested.

[0013] The sample to be tested was obtained and subjected to protein hydrolysis to obtain a hydrolysate containing free amino acids, including:

[0014] The sample to be tested is subjected to chemical hydrolysis and enzymatic hydrolysis in sequence. The chemical hydrolysis and enzymatic hydrolysis are carried out in a hydrolysis unit equipped with an ion exchange membrane group, which is used to remove interfering ions generated during the hydrolysis process.

[0015] The pH value and conductivity of the hydrolysis products during the hydrolysis process are detected, and the degree of hydrolysis is determined based on the pH value and conductivity.

[0016] Adjust the hydrolysis treatment conditions based on the degree of hydrolysis until the pH value and conductivity of the hydrolysis products reach the preset range;

[0017] The hydrolysis product is subjected to ion exchange treatment through the ion exchange membrane assembly to obtain a hydrolysate containing free amino acids.

[0018] The hydrolysate and derivatizing reagent are mixed and then sequentially passed through multiple reaction zones arranged in series for online derivatization. Each reaction zone maintains a different pH environment for amino acids with different dissociation constants, resulting in a derivatization reaction solution containing amino acid derivatives, including:

[0019] The pH value in the hydrolysate was measured using an electrochemical detector, and the conductivity of the hydrolysate was measured using a conductivity detector. Based on the pH value and conductivity, an acid-base characteristic curve of the hydrolysate was generated.

[0020] The free amino acids in the hydrolysate are classified according to the acid-base characteristic curves, the pH environment required for the free amino acids in the multi-stage reaction zone set in series is determined, and the amount of hydrolysate and derivatizing reagent is calculated.

[0021] The hydrolysate and the derivatizing reagent are delivered to the eddy mixing device according to the specified dosage. The eddy mixing device uses eddy shear force to fully mix the hydrolysate and the derivatizing reagent to form a mixed reaction solution.

[0022] The buffer solution is delivered to multiple reaction zones arranged in series, so that the multiple reaction zones form a pH environment adapted to different free amino acids;

[0023] The mixed reaction solution is subjected to an online derivatization reaction through multiple reaction zones arranged in series. The pH changes of the multiple reaction zones are monitored in real time using a detector. The amount of buffer solution replenished is adjusted according to the pH changes to maintain the pH environment of the multiple reaction zones, thereby obtaining a derivatization reaction solution containing amino acid derivatives.

[0024] An online detection unit is installed at the outlet of the multi-stage reaction zone to monitor the concentration of amino acid derivatives generated. Based on the generated concentration, the derivatizing reagent supply rate and the reaction zone temperature are adjusted in real time to obtain a mobile phase containing amino acid derivatives, comprising:

[0025] The fluorescence signal of amino acid derivatives at the outlet of the multi-stage reaction zone is detected, and the temperature and pressure data of the multi-stage reaction zone are recorded. The concentration of amino acid derivatives generated is calculated based on the fluorescence signal, temperature data and pressure data.

[0026] The formation pattern of amino acid derivatives is determined based on the correspondence between the formation concentrations under different temperature and pressure data, and the degree of completion of the derivatization reaction is calculated based on the formation pattern.

[0027] The supply rate of the derivatizing reagent and the temperature of the reaction zone are adjusted by calculating the completion rate, thereby controlling the delivery speed of the derivatizing reagent and the heating power of the reaction zone.

[0028] The concentration of amino acid derivatives after adjustment is monitored. When the adjusted concentration meets the chromatographic separation conditions, a mobile phase containing amino acid derivatives is obtained. The mobile phase contains the amino acid derivatives that have completed the derivatization reaction and the mobile phase solvent.

[0029] The mobile phase containing amino acid derivatives was subjected to chromatographic separation to obtain amino acid derivative components separated in order of elution time, including:

[0030] The polarity value of amino acid derivatives in the mobile phase is detected, and the polarity range of amino acid derivatives is divided according to the polarity value. Based on the polarity range, the range of change of the mobile phase gradient and the stationary phase material of the chromatographic column are determined, and a gradient elution program is generated.

[0031] The gradient elution program is executed, the ultraviolet detection signal at the column outlet is collected, and the retention time difference and peak width ratio of adjacent amino acid derivatives are calculated.

[0032] The resolution values ​​of adjacent chromatographic peaks are calculated based on the retention time difference and peak width ratio, and the rate of change of the gradient elution program is adjusted based on the resolution values.

[0033] The adjusted gradient elution program is executed, and the ultraviolet detection signal at the column outlet is monitored. When the ultraviolet detection signal indicates that the chromatographic peak separation is complete, the amino acid derivative components separated in the order of elution time are obtained.

[0034] Based on the elution time window of the amino acid derivative components, the ionization polarity mode of the ion source is switched during mass spectrometry detection to ensure that the amino acid derivative components are detected in the mode with optimal ionization efficiency. The obtained mass spectrometry response signal includes:

[0035] The ultraviolet detection signal and ion current signal of the amino acid derivative component are collected. The elution time segment of the amino acid derivative component is determined by the peak position of the ultraviolet detection signal and ion current signal, and the elution time segment is set as the elution time window.

[0036] The amino acid derivative components are detected in positive and negative ionization modes according to the elution time window. Mass spectrometry signals are obtained through the positive and negative ionization modes. The signal-to-noise ratio and peak shape ratio of the mass spectrometry signals are calculated. The optimal ionization efficiency mode is determined based on the signal-to-noise ratio and peak shape ratio.

[0037] The optimal ionization efficiency mode is combined with the elution time window to generate an ionization polarity switching command, which is used to control the ionization polarity mode of the ion source during mass spectrometry detection.

[0038] The response signal output by the mass spectrometer detector is monitored, and when the response signal indicates that the amino acid derivative component has been detected, the mass spectrometer response signal of the amino acid derivative component is acquired.

[0039] Substituting the mass spectrometry response signal into a preset standard curve, the amino acid concentration values ​​corresponding to the amino acid derivative components in the sample to be tested are calculated, including:

[0040] The response intensity and response deviation of the amino acid derivative component in the mass spectrometry response signal are collected at multiple mass-to-charge ratios. The quantitative mass-to-charge ratio is selected according to the ratio of the response intensity to the response deviation value, and the response signal at the quantitative mass-to-charge ratio is obtained.

[0041] Standard response signals of amino acid derivative standard solutions of known concentrations at the given quantitative mass-to-charge ratio are collected, and a standard curve is established between the standard response signal and the concentration. The response signal is then normalized using the standard curve to obtain the standardized response value.

[0042] Substitute the standardized response value into the standard curve, calculate the conversion coefficient between the standardized response value and the concentration, and calculate the amino acid concentration value using the conversion coefficient.

[0043] Verify the accuracy of the amino acid concentration value. When the accuracy meets the measurement requirements, obtain the amino acid concentration value corresponding to the amino acid derivative component in the sample to be tested.

[0044] One technical solution provided in this embodiment of the invention is an electronic device, including: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the steps in any of the aforementioned methods.

[0045] One technical solution provided in this embodiment of the invention is a computer-readable storage medium storing computer program instructions, which, when executed by a processor, implement the steps in any of the aforementioned methods.

[0046] This invention, by setting up multiple reaction zones and maintaining different pH environments, can provide optimal reaction conditions for amino acids with different dissociation constants, thereby improving the conversion efficiency of derivatization reactions. Utilizing an online detection unit to monitor the concentration of derivatized products in real time and dynamically adjust reaction conditions enables precise control of the derivatization reaction, ensuring the stability of the derivatized products. The tandem reaction zone structure avoids manual operation steps, improving automation and reducing human error. Switching the ion source ionization polarity mode through the elution time window allows for the detection of each amino acid derivative under optimal ionization conditions, enhancing detection sensitivity. The overall solution achieves fully automated online operation of the entire amino acid detection process, significantly improving detection efficiency and result accuracy while reducing detection costs, demonstrating significant practical value. Attached Figure Description

[0047] Figure 1 A flowchart of an online pre-column derivatization coupled liquid chromatography-mass spectrometry method for amino acid detection provided in an embodiment of the present invention;

[0048] Figure 2 This is a flowchart illustrating the online monitoring and feedback control process for amino acid derivatization reactions according to an embodiment of the present invention.

[0049] Figure 3 This is a schematic diagram comparing the chromatographic separation effects before and after adjusting the gradient elution program in an embodiment of the present invention. Detailed Implementation

[0050] like Figure 1 As shown, Figure 1 A flowchart of an online pre-column derivatization coupled liquid chromatography-mass spectrometry (LC-MS) method for amino acid detection provided in an embodiment of the present invention is shown. The method includes the following steps:

[0051] The sample to be tested was obtained and subjected to protein hydrolysis to obtain a hydrolysate containing free amino acids;

[0052] After the hydrolysate is mixed with the derivatizing reagent, it is sequentially passed through multiple reaction zones arranged in series for online derivatization. Each reaction zone maintains a different pH environment for amino acids with different dissociation constants, thus obtaining a derivatization reaction solution containing amino acid derivatives.

[0053] An online detection unit is set at the outlet of the multi-stage reaction zone to monitor the concentration of amino acid derivatives generated. The supply rate of the derivatizing reagent and the temperature of the reaction zone are adjusted in real time according to the concentration to obtain a mobile phase containing amino acid derivatives.

[0054] The mobile phase containing amino acid derivatives was subjected to chromatographic separation to obtain amino acid derivative components separated in order of elution time.

[0055] Based on the elution time window of the amino acid derivative components, the ionization polarity mode of the ion source is switched during the mass spectrometry detection process so that the amino acid derivative components are detected in the mode with the optimal ionization efficiency, and the mass spectrometry response signal is obtained.

[0056] Substitute the mass spectrometry response signal into the preset standard curve to calculate the amino acid concentration value corresponding to the amino acid derivative component in the sample to be tested.

[0057] The sample to be tested was obtained and subjected to protein hydrolysis to obtain a hydrolysate containing free amino acids, including:

[0058] The sample to be tested is subjected to chemical hydrolysis and enzymatic hydrolysis in sequence. The chemical hydrolysis and enzymatic hydrolysis are carried out in a hydrolysis unit equipped with an ion exchange membrane group, which is used to remove interfering ions generated during the hydrolysis process.

[0059] The pH value and conductivity of the hydrolysis products during the hydrolysis process are detected, and the degree of hydrolysis is determined based on the pH value and conductivity.

[0060] Adjust the hydrolysis treatment conditions based on the degree of hydrolysis until the pH value and conductivity of the hydrolysis products reach the preset range;

[0061] The hydrolysis product is subjected to ion exchange treatment through the ion exchange membrane assembly to obtain a hydrolysate containing free amino acids.

[0062] The sample to be tested can be a protein-containing substance such as food, biological products, or pharmaceutical products. After obtaining the sample, pretreatment is required, including freeze-drying and grinding, to ensure the sample reaches a homogeneous state. The ground sample should be sieved through a 100-mesh sieve to ensure the particle size is suitable for the subsequent hydrolysis process. After sample pretreatment, weigh an appropriate amount of sample, typically 50-200 mg, and place it in a dedicated hydrolysis tube.

[0063] The hydrolysis unit consists of a hydrolysis reaction chamber, an ion exchange membrane assembly, and a detection module. The hydrolysis reaction chamber, made of high-temperature and corrosion-resistant material, has a volume of 10-20 mL and a sealed structure to prevent sample evaporation during hydrolysis. The ion exchange membrane assembly, installed within the hydrolysis reaction chamber, comprises cation exchange membranes and anion exchange membranes. The cation exchange membranes utilize sulfonic acid functional groups with an exchange capacity of 1.2-2.0 meq / g; the anion exchange membranes utilize quaternary ammonium functional groups with an exchange capacity of 1.0-1.8 meq / g. The ion exchange membranes are arranged in parallel or ring formations to form ion-capturing regions, used to remove interfering ions generated during hydrolysis, such as metal ions and chloride ions.

[0064] Chemical hydrolysis is the first stage of protein hydrolysis. A 6 mol / L hydrochloric acid solution is added to the hydrolysis reaction chamber at a solution-to-sample ratio of 20:1, and the chamber is then sealed. The mixture is heated to 110°C and hydrolyzed at this temperature for 12-24 hours. Chemical hydrolysis breaks most peptide bonds in the protein, initially hydrolyzing it into polypeptides and some amino acids. Special attention must be paid to amino acids such as tryptophan, methionine, and glutamine, which are easily damaged by strong acids, during this stage. After chemical hydrolysis, the mixture is cooled to room temperature and then neutralized at pH by adding a 2 mol / L sodium hydroxide solution to adjust the pH to 6.5-7.0.

[0065] Enzymatic hydrolysis, as the second stage of hydrolysis, targets peptide structures that are difficult to completely break down through chemical hydrolysis. A mixture of proteases, including trypsin, papain, and pepsin, is prepared in a 1:1:1 ratio, with a total enzyme content to sample mass ratio of 1:50. The enzymatic hydrolysis reaction is controlled at 37℃ and maintained for 4-8 hours. During the enzymatic hydrolysis process, the pH is maintained within the range of 6.5-7.5, and pH is adjusted by adding 0.1 mol / L phosphate buffer if necessary. Enzymatic hydrolysis can further hydrolyze peptides, increasing the release of free amino acids, especially for amino acids such as asparagine and glutamine, which are easily denatured during chemical hydrolysis; enzymatic hydrolysis can improve their detection rate.

[0066] The hydrolysis unit incorporates a pH electrode and a conductivity sensor to monitor the pH and conductivity of the solution in real time during hydrolysis. When hydrolysis is complete, the solution pH should stabilize within the range of 6.0-7.5, and the conductivity should reach 8-15 mS / cm. Hydrolysis is considered to have reached a steady state when the pH or conductivity fluctuation is less than 3% for 30 consecutive minutes. Incomplete hydrolysis is characterized by unstable pH or a continuous increase in conductivity, requiring adjustment of the hydrolysis conditions.

[0067] Hydrolysis conditions are adjusted based on monitoring data. If the pH value is below 6.0, it indicates an overly acidic environment, and 0.1 mol / L sodium hydroxide solution can be added for adjustment. If the pH value is above 7.5, it indicates an overly alkaline environment, and 0.1 mol / L hydrochloric acid solution can be added for adjustment. Low conductivity indicates incomplete hydrolysis; the hydrolysis time can be extended by 1-2 hours or the temperature increased by 2-5°C. High conductivity indicates possible over-hydrolysis or impurity interference; the temperature needs to be lowered or the ion exchange membrane assembly replaced. During adjustment, after each parameter change, observe for 15-30 minutes before deciding whether further adjustments are needed.

[0068] The hydrolysis products are treated using an ion-exchange membrane array. The cation exchange membrane adsorbs the metal cations generated during hydrolysis, while the anion exchange membrane adsorbs anions such as chloride ions. The ion exchange process is carried out at room temperature, with a flow rate controlled at 1-2 mL / min. To improve exchange efficiency, oscillation assistance can be used at a frequency of 60-120 times / min. After ion exchange treatment, the pH of the hydrolysate should be between 5.5 and 7.5, and the conductivity should decrease to 2-5 mS / cm, indicating that interfering ions have been effectively removed.

[0069] After the hydrolysate is prepared, post-processing is required. The hydrolysate should be filtered through a 0.22 μm filter membrane to remove insoluble impurities and any remaining enzymes or proteins. The filtered hydrolysate can be used directly for amino acid analysis or it can be concentrated under vacuum at a temperature not exceeding 45°C to prevent thermal degradation of the amino acids. The concentrated hydrolysate should be stored at -20°C in a dark place for up to 7 days.

[0070] For example, 100 mg of sample was pretreated and placed in a hydrolysis reaction chamber. 2 mL of 6 mol / L hydrochloric acid solution was added, and the mixture was sealed and hydrolyzed at 110°C for 18 hours. After cooling, the pH was adjusted to 6.8 with 2 mol / L sodium hydroxide. 2 mg of a mixed enzyme preparation was added, and enzymatic hydrolysis was performed at 37°C for 6 hours. During the later stages of hydrolysis, the pH stabilized at 6.9 ± 0.1, and the conductivity was 12.5 mS / cm with fluctuations of less than 2% within 30 minutes. After treatment with an ion exchange membrane, the hydrolysate had a pH of 6.7 and a conductivity reduced to 3.2 mS / cm. Filtering through the membrane yielded a transparent hydrolysate.

[0071] This invention achieves complete protein hydrolysis through the combined application of chemical and enzymatic hydrolysis, ensuring the intact release of all amino acids. The introduction of an ion-exchange membrane array removes interfering ions, significantly reducing the impact of impurities on subsequent analysis. Real-time monitoring of pH and conductivity parameters establishes a precise assessment system for the degree of hydrolysis, avoiding the blind spots inherent in traditional hydrolysis methods. Dynamic adjustment of hydrolysis conditions based on monitoring results enables precise control of the hydrolysis process, resulting in more complete amino acid release and more accurate analytical results. The overall process optimizes protein hydrolysis efficiency, improves the sensitivity and reliability of amino acid detection, and provides strong technical support for quality control in the food and pharmaceutical industries.

[0072] The hydrolysate and derivatizing reagent are mixed and then sequentially passed through multiple reaction zones arranged in series for online derivatization. Each reaction zone maintains a different pH environment for amino acids with different dissociation constants, resulting in a derivatization reaction solution containing amino acid derivatives, including:

[0073] The pH value in the hydrolysate was measured using an electrochemical detector, and the conductivity of the hydrolysate was measured using a conductivity detector. Based on the pH value and conductivity, an acid-base characteristic curve of the hydrolysate was generated.

[0074] The free amino acids in the hydrolysate are classified according to the acid-base characteristic curves, the pH environment required for the free amino acids in the multi-stage reaction zone set in series is determined, and the amount of hydrolysate and derivatizing reagent is calculated.

[0075] The hydrolysate and the derivatizing reagent are delivered to the eddy mixing device according to the specified dosage. The eddy mixing device uses eddy shear force to fully mix the hydrolysate and the derivatizing reagent to form a mixed reaction solution.

[0076] The buffer solution is delivered to multiple reaction zones arranged in series, so that the multiple reaction zones form a pH environment adapted to different free amino acids;

[0077] The mixed reaction solution is subjected to an online derivatization reaction through multiple reaction zones arranged in series. The pH changes of the multiple reaction zones are monitored in real time using a detector. The amount of buffer solution replenished is adjusted according to the pH changes to maintain the pH environment of the multiple reaction zones, thereby obtaining a derivatization reaction solution containing amino acid derivatives.

[0078] An electrochemical detector and a conductivity detector simultaneously receive the hydrolysate sample. The electrochemical detector uses a glass composite pH electrode with a measurement range of pH 0-14, an accuracy of ±0.02 pH units, and an electrode response time of less than 10 seconds. The measured pH value is transmitted to the data processing unit as a digital signal. The conductivity detector uses a four-electrode measurement unit with a measurement range of 0-200 mS / cm, an accuracy of ±1%, and a temperature compensation range of 5-50°C. The conductivity value is also transmitted to the data processing unit. The data processing unit records the pH and conductivity trends at a sampling frequency of 5 data points per second, acquiring a dataset containing at least 300 valid measurement points.

[0079] The data processing unit generates an acid-base characteristic curve for the hydrolysate based on the collected pH and conductivity data. This curve describes the conductivity response characteristics of the hydrolysate during pH changes, and its shape reflects the distribution of amino acids with different dissociation constants in the hydrolysate. Data processing employs a moving average algorithm to smooth the raw data, with a window width of 5 data points to remove noise. Subsequently, a piecewise linear fitting method is used to determine the inflection points of the curve. These inflection points correspond to the dissociation pH thresholds for different amino acid types, and the inflection point criterion is a point where the slope change rate is greater than 30%. The fitting accuracy requires the sum of squared residuals to be less than 5% of the total variation to ensure the representativeness of the characteristic curve.

[0080] Acidic amino acids, including aspartic acid and glutamic acid, have a pKa value below 4.0, making them suitable for derivatization reactions within the pH range of 3.0-4.5. Neutral amino acids, such as alanine and valine, have a pKa value between 5.0 and 6.5, making them suitable for derivatization reactions within the pH range of 5.5-7.0. Basic amino acids, such as lysine and arginine, have a pKa value above 7.5, making them suitable for derivatization reactions within the pH range of 8.0-9.5. Based on the piecewise fitting results of the characteristic curves, the proportion of each amino acid in the hydrolysate is calculated, and this proportion is used to determine the amount of derivatizing reagent. The derivatizing reagent is a mixture of phthalaldehyde and 2-mercaptoethanol, with phthalaldehyde at a concentration of 10 mM and 2-mercaptoethanol at a concentration of 20 mM. The ratio of hydrolysate to derivatizing reagent is typically 1:2 to 1:5, with the specific amount calculated based on the total amount of amino acids in the hydrolysate, ensuring a derivatizing reagent excess of at least 50% to guarantee complete reaction.

[0081] A vortex mixing device is used to achieve efficient mixing of hydrolysate and derivatizing reagent. The device consists of a microchannel mixing unit and an ultrasonic-assisted system. The microchannel mixing unit adopts a labyrinthine structure with a channel width of 100-200 μm, a depth of 150-250 μm, and a total length of 10-15 cm. The hydrolysate and derivatizing reagent are delivered separately by two micro-injection pumps at flow rates ranging from 0.1-2.0 mL / min and a pressure limit of 20 MPa. After the two liquids converge at a T-joint, they enter the labyrinthine microchannel. The microchannel incorporates multiple 90° turns and serrated structures, generating strong vortex shear forces. Under these shear forces, the liquids form cascaded flows, significantly increasing the boundary area and shortening the molecular diffusion distance to the micrometer level, with a mixing time of less than 1 second. The ultrasonic-assisted system operates at a frequency of 40 kHz and a power density of 0.5 W / cm². 2 The intermittent working mode is 5 seconds on and 2 seconds off, which further enhances the mixing effect and forms a uniform mixed reaction liquid.

[0082] The buffer solution system provides a stable pH environment for the series reaction zones. The buffer solutions are stored in three independent reservoirs: an acetate buffer (pH 4.0), a phosphate buffer (pH 7.0), and a borax buffer (pH 9.0), each with a concentration of 50 mM. These three buffer solutions are delivered to their respective reaction zones via independent high-precision plunger pumps at flow rates ranging from 0.05 to 0.5 mL / min, with an accuracy of ±0.5%. The series-connected multi-stage reaction zones consist of three independent reaction units, each with a volume of 0.5–1.0 mL, constructed of inert fluoropolymer or titanium alloy, with a temperature tolerance range of 10–100°C and a pressure resistance of at least 10 MPa. Each reaction unit incorporates a temperature sensor and a pH microelectrode to monitor the reaction environment in real time. The first reaction zone maintains a pH of 3.5–4.5 for the derivatization of acidic amino acids; the second reaction zone maintains a pH of 6.0–7.0 for the derivatization of neutral amino acids; and the third reaction zone maintains a pH of 8.5–9.5 for the derivatization of basic amino acids.

[0083] After the mixed reaction solution enters a series of multi-stage reaction zones, differentiated derivatization reactions occur under different pH conditions. Each reaction zone is equipped with a micro-turbo stirrer with a rotation speed of 300-500 rpm to ensure reaction uniformity. The temperature of the first reaction zone is controlled at 40±1°C, and the reaction time is 60-90 s; the temperature of the second reaction zone is controlled at 50±1°C, and the reaction time is 90-120 s; the temperature of the third reaction zone is controlled at 60±1°C, and the reaction time is 120-180 s. A pH microelectrode within the reaction zone continuously monitors pH changes, sampling at a frequency of 5 s / time. A buffer replenishment mechanism is triggered when the pH fluctuation exceeds ±0.2 units. The buffer replenishment amount is calculated using a proportional-integral control algorithm, with a response time of less than 3 s, a stabilization time of less than 15 s, and a pH control accuracy of ±0.1 units. During the reaction, amino acids combine with o-phthalaldehyde and 2-mercaptoethanol to form isoindole derivatives with high fluorescence intensity. Different types of amino acids complete the derivatization reaction under their respective optimal pH conditions.

[0084] For example, 1 mL of the hydrolysate sample was taken, and the pH value was measured to be 6.8 and the conductivity to be 12.5 mS / cm using an electrochemical detector. The generated acid-base characteristic curve showed three distinct inflection points, corresponding to pH 4.2, pH 6.5, and pH 8.7, respectively. Based on this, the amino acids in the hydrolysate were classified into three categories, and the total amount of derivatizing reagent required was calculated to be 3 mL. The hydrolysate and derivatizing reagent were mixed at a ratio of 1:3 using a vortex mixer to form a mixed reaction solution, with the flow rate set at 1.2 mL / min. Buffer solutions were introduced into the three reaction zones at flow rates of 0.2 mL / min, 0.3 mL / min, and 0.25 mL / min, respectively, to establish a pH gradient environment. The mixed reaction solution was passed through the three reaction zones sequentially. In the first reaction zone, the pH was stabilized at 4.2 ± 0.1 for 75 s; in the second reaction zone, the pH was stabilized at 6.7 ± 0.1 for 105 s; and in the third reaction zone, the pH was stabilized at 9.1 ± 0.1 for 150 s. pH monitoring showed that the pH in the second reaction zone dropped to 6.5 after 60 seconds of reaction. The buffer flow rate was automatically increased to 0.35 mL / min, and the pH recovered to 6.7 within 10 seconds. The conversion rate of amino acid derivatives in the derivatized reaction solution obtained throughout the process reached over 98.5%.

[0085] This invention achieves differentiated online derivatization reactions of amino acids with different dissociation constants by establishing a multi-stage pH gradient environment in series reaction zones. Compared with traditional batch derivatization methods under a single pH environment, this significantly improves the generation efficiency and detection sensitivity of amino acid derivatives. Especially for complex biological samples containing acidic, neutral, and basic amino acids simultaneously, the differentiated pH environment can maximize the preservation of the structural integrity of various amino acids, reduce cross-interference, and improve detection accuracy.

[0086] An online detection unit is installed at the outlet of the multi-stage reaction zone to monitor the concentration of amino acid derivatives generated. Based on the generated concentration, the derivatizing reagent supply rate and the reaction zone temperature are adjusted in real time to obtain a mobile phase containing amino acid derivatives, comprising:

[0087] The fluorescence signal of amino acid derivatives at the outlet of the multi-stage reaction zone is detected, and the temperature and pressure data of the multi-stage reaction zone are recorded. The concentration of amino acid derivatives generated is calculated based on the fluorescence signal, temperature data and pressure data.

[0088] The formation pattern of amino acid derivatives is determined based on the correspondence between the formation concentrations under different temperature and pressure data, and the degree of completion of the derivatization reaction is calculated based on the formation pattern.

[0089] The supply rate of the derivatizing reagent and the temperature of the reaction zone are adjusted by calculating the completion rate, thereby controlling the delivery speed of the derivatizing reagent and the heating power of the reaction zone.

[0090] The concentration of amino acid derivatives after adjustment is monitored. When the adjusted concentration meets the chromatographic separation conditions, a mobile phase containing amino acid derivatives is obtained. The mobile phase contains the amino acid derivatives that have completed the derivatization reaction and the mobile phase solvent.

[0091] like Figure 2 The diagram illustrates the online monitoring and feedback control flowchart for the amino acid derivatization reaction in this embodiment. Specifically, the online detection unit consists of a fiber optic fluorescence detector, a thermocouple temperature sensor, and a pressure sensor. The fiber optic fluorescence detector is installed at the outlet of each reaction zone, using an ultraviolet light source with an excitation wavelength of 340 nm and a detection wavelength range of 425-480 nm, corresponding to the fluorescence emission range of the amino acid derivative. The detector uses a photomultiplier tube as the receiving element, with a sensitivity of up to 1 ng / mL and a linear range of 1-500 ng / mL. The thermocouple temperature sensor is directly inserted into the fluid in the reaction zone, with a temperature measurement range of 0-100℃ and an accuracy of ±0.2℃. The pressure sensor is installed at the inlet and outlet of the reaction zone, with a measurement range of 0-20 MPa and an accuracy of ±0.5%. The data acquisition frequency of these three sensors is 5 Hz, ensuring real-time monitoring of the reaction status.

[0092] When amino acids react with phthalaldehyde-mercapto-reagent to generate fluorescent products, the fluorescence intensity is directly proportional to the concentration of the derivative. The fluorescence signal acquired by the fiber optic fluorescence detector is converted from analog to digital and then transmitted to the data processing unit. The data processing unit uses a built-in standard curve conversion algorithm to convert the fluorescence signal into concentration values. This standard curve is established by pre-injecting a series of standards with known concentrations, covering 20 concentration points in the range of 0.5-100 μg / mL, with a correlation coefficient greater than 0.995. To eliminate the influence of temperature on fluorescence intensity, a built-in temperature correction function is used. Within the range of 25-65℃, the fluorescence intensity decreases by approximately 6.5% for every 5℃ increase; real-time correction is performed based on this rule.

[0093] The concentration was calculated using a corrected integral algorithm, averaging the fluorescence signal over 10 consecutive seconds and combining it with flow rate parameters to determine the mass concentration. The calculation formula considered factors such as flow rate, optical path length, and molecular weight. When pressure changed, a fluid volume correction factor corresponding to the pressure was obtained through a lookup table to correct the concentration calculation results. When the pressure increased from atmospheric pressure to 5 MPa, the fluid volume compressed by approximately 2.3%, requiring a 2.3% correction to the concentration calculation. The average, maximum, and minimum concentrations were recorded every minute to generate a concentration-time curve.

[0094] The formation patterns of amino acid derivatives were determined using a data fitting method, collecting continuous monitoring data for at least 5 minutes, including concentration trends under different temperature and pressure conditions. The data processing unit employed multiple regression analysis to establish a model relating temperature, pressure, reaction time, and product concentration. This model includes temperature, pressure, and time terms and their interactions, and can predict product concentrations under different combinations of conditions. For aspartic acid derivatives, the reaction rate increased by approximately 70% when the temperature increased from 40℃ to 50℃; while for lysine derivatives, the same temperature change increased the reaction rate by approximately 55%.

[0095] The completion rate of the derivatization reaction is defined as the percentage of the actual measured concentration relative to the theoretical maximum concentration. The theoretical maximum concentration is calculated using the total amount of amino acids in the hydrolysate, the conversion efficiency of the derivatizing reagent, and the dilution factor. A completion rate threshold of 90% is set; the reaction is considered to have reached a steady state when the detected concentration of the amino acid derivative reaches more than 90% of the theoretical maximum and the concentration change is less than 3% for 30 consecutive seconds. For complex samples, the completion rate of the major amino acid species is monitored, ensuring that the completion rate of at least eight essential amino acids exceeds the threshold.

[0096] The derivatization reagent supply rate is adjusted based on a completion rate feedback control algorithm. When the completion rate is below 80%, the supply rate is increased by 10-30%; when the completion rate is between 80-90%, it is increased by 5-10%; and when the completion rate exceeds 95%, the current supply rate remains unchanged. The derivatization reagent supply device uses a precision plunger pump with a flow rate range of 0.01-5 mL / min and an adjustment accuracy of 0.01 mL / min. To avoid background interference caused by excessive reagent, the maximum supply of derivatization reagent is set to no more than three times the theoretical requirement.

[0097] The reaction zone temperature is controlled using a proportional-integral-derivative (PID) control strategy. When the completion rate falls below the target value, the temperature adjustment range is calculated. Generally, a 5% reduction in completion rate requires an increase in reaction temperature of 2-3°C. The temperature adjustment range is limited to 35-65°C, with a heating rate controlled below 1°C / minute to avoid localized overheating. Each reaction zone is equipped with an independent heating element and heat dissipation system, with a heating power range of 0-100W and a control accuracy of ±0.5°C. To prevent derivative degradation caused by temperature fluctuations, a temperature gradient limit is set, ensuring that the temperature change between adjacent time points does not exceed 2°C.

[0098] The adjusted reaction state should be continuously monitored for at least 2 minutes to confirm that the generated concentration is stable and meets the chromatographic separation requirements. The chromatographic separation conditions require the total concentration of amino acid derivatives in the mobile phase to be in the range of 5-50 μg / mL, with a relative standard deviation of less than 5%, and the pH value to be stable between 6.5 and 7.5. When the monitoring results meet the above conditions, the mobile phase containing amino acid derivatives is introduced into the subsequent chromatographic separation unit. The mobile phase mainly contains the derivatized amino acid fluorescent derivatives and solvent components suitable for chromatographic separation, consisting of phosphate buffer and organic regulators.

[0099] Taking glutamate derivatization as an example, when the initial derivatizing reagent supply rate was 0.5 mL / min and the reaction zone temperature was 45℃, the detected concentration of glutamate derivative was 15 μg / mL, with a completion rate of 75%. The derivatizing reagent supply rate was automatically increased to 0.65 mL / min, and the reaction zone temperature was simultaneously increased to 50℃. After 60 seconds of adjustment, the detected concentration of glutamate derivative rose to 19 μg / mL, achieving a completion rate of 95%, with concentration fluctuations less than 2%, meeting the chromatographic separation requirements. At this point, the concentration of glutamate derivative in the mobile phase was stable, and the mobile phase pH was 7.2, suitable for subsequent chromatographic separation steps.

[0100] This invention utilizes an online detection unit to monitor and provide feedback on the concentration of amino acid derivatives in real time, enabling precise control and optimization of the derivatization process. Compared to traditional batch derivatization methods with fixed parameters, this invention allows for dynamic adjustment of reaction conditions based on different sample characteristics, improving derivatization efficiency and stability. The multi-parameter synergistic control strategy effectively addresses the significant differences in derivatization efficiency among different amino acids in complex samples, reducing analytical errors. The combination of real-time monitoring and intelligent control not only improves the accuracy and sensitivity of amino acid detection but also shortens analysis time and reduces reagent consumption.

[0101] The mobile phase containing amino acid derivatives was subjected to chromatographic separation to obtain amino acid derivative components separated in order of elution time, including:

[0102] The polarity value of amino acid derivatives in the mobile phase is detected, and the polarity range of amino acid derivatives is divided according to the polarity value. Based on the polarity range, the range of change of the mobile phase gradient and the stationary phase material of the chromatographic column are determined, and a gradient elution program is generated.

[0103] The gradient elution program is executed, the ultraviolet detection signal at the column outlet is collected, and the retention time difference and peak width ratio of adjacent amino acid derivatives are calculated.

[0104] The resolution values ​​of adjacent chromatographic peaks are calculated based on the retention time difference and peak width ratio, and the rate of change of the gradient elution program is adjusted based on the resolution values.

[0105] The adjusted gradient elution program is executed, and the ultraviolet detection signal at the column outlet is monitored. When the ultraviolet detection signal indicates that the chromatographic peak separation is complete, the amino acid derivative components separated in the order of elution time are obtained.

[0106] The polarity of amino acid derivatives in the mobile phase was determined using high-performance liquid chromatography (HPLC) retention factor assay. The mobile phase sample was injected into an analytical column equipped with a diode array detector. A methanol-water system was used as the test mobile phase, and the retention times of the amino acid derivatives were measured under different methanol ratios. The retention factor was calculated based on the retention time; a higher retention factor value indicates stronger polarity. The amino acid derivatives contained in the mobile phase sample typically fall into three categories: acidic, neutral, and basic. The retention factor values ​​for each category of amino acid derivatives are distributed in different ranges. Acidic amino acid derivatives, such as aspartic acid and glutamic acid, have retention factors ranging from 0.8 to 2.5; neutral amino acid derivatives, such as alanine and valine, have retention factors ranging from 2.5 to 5.0; and basic amino acid derivatives, such as lysine and arginine, have retention factors ranging from 5.0 to 10.0. Polarity intervals were defined based on the retention factor values, generating a polarity distribution map. This polarity distribution map is a two-dimensional rectangular coordinate graph, with the horizontal axis representing the retention factor value and the vertical axis representing the response intensity or relative content of each amino acid derivative under the test conditions. Once the polarity range is determined, a chromatographic column stationary phase material that matches the polarity characteristics of the amino acid derivative is selected.

[0107] For amino acids derived from phthalaldehyde-mercaptoethanol, reversed-phase chromatography provides good separation, typically using a C18 stationary column. The column is 150 mm long, 4.6 mm inner diameter, with a packing particle size of 3 μm, a pore size of 100 μm, a carbon loading of 15%, an end-capping efficiency greater than 95%, and a theoretical plate number of at least 15,000 / m. The mobile phase gradient range is determined based on the polarity distribution: Phase A is 10 mmol / L sodium dihydrogen phosphate buffer, pH adjusted to 6.8; Phase B is acetonitrile containing 0.1% trifluoroacetic acid. The gradient elution program design considers three key points: the initial B phase ratio, the final B phase ratio, and the gradient curve shape. The initial B phase ratio is set to 10% to ensure that highly polar amino acid derivatives react sufficiently with the mobile phase without being directly eluted; the final B phase ratio is set to 90% to ensure that low-polarity amino acid derivatives are completely eluted; a three-segment linear gradient is selected to correspond to the elution requirements of the three types of amino acid derivatives.

[0108] During the gradient elution process, a UV detector is used to acquire the signal at the column outlet. The UV detector is set with a dominant wavelength of 338 nm, a reference wavelength of 450 nm, a sampling frequency of 10 Hz, a detection sensitivity of 0.001 AUFS, and a linear range of 0.001–2.000 AU. The chromatogram data undergoes baseline correction and peak identification processing, automatically marking the start, peak apex, and end points of each peak. For each identified peak, the retention time, peak height, peak area, and half-maximum width (WHM) are calculated. The retention time difference between adjacent peaks is obtained by subtracting the retention times at the peak apex of adjacent peaks; the peak width ratio is calculated by summing the WHMs of two adjacent peaks and taking the average.

[0109] The resolution of adjacent chromatographic peaks is an important indicator for evaluating separation efficiency. It is calculated by dividing the difference in retention time between adjacent peaks by the average half-peak width (HWHM) of the two peaks. A resolution greater than 1.5 indicates complete separation; a resolution between 1.0 and 1.5 indicates partial overlap but still quantifiable; and a resolution less than 1.0 indicates severe overlap making accurate quantification impossible. The gradient elution program is adjusted based on the resolution. The adjustment principles are as follows: when the resolution is greater than 2.0, the gradient rate can be increased appropriately, shortening the analysis time; when the resolution is between 1.5 and 2.0, the current gradient rate remains unchanged; when the resolution is between 1.0 and 1.5, the gradient rate is reduced by 10-20%; and when the resolution is less than 1.0, the gradient rate is reduced by 30-50%, while considering adding an isocratic elution zone. Gradient rate adjustment is achieved by modifying the gradient time; for example, extending a 10-minute gradient to 12 minutes reduces the gradient rate by 20%.

[0110] The adjusted gradient elution program was executed again, and the UV detection signal at the column outlet was monitored continuously. When the resolution of all target amino acid derivative peaks on the chromatogram was greater than 1.2, and the peak shape symmetry factor was within the range of 0.8-1.2, the chromatographic peak separation was considered complete. After chromatographic separation, each amino acid derivative component was eluted sequentially according to its retention behavior in the chromatographic column, typically in the following order: acidic amino acid derivatives, neutral amino acid derivatives, and basic amino acid derivatives. Each component corresponds to a chromatographic peak, and its retention time, peak area, and peak height data were recorded using a chromatography workstation for subsequent mass spectrometry analysis and quantitative calculations.

[0111] After derivatization, a mixed amino acid standard solution was initially programmed with the following gradient: 0-5 minutes, phase B 10-30%; 5-15 minutes, phase B 30-70%; 15-20 minutes, phase B 70-90%. After executing this program, the resolution between glutamic acid and aspartic acid derivatives was found to be 0.85, which did not meet the separation requirements. The retention time difference between glutamic acid and aspartic acid derivatives was calculated to be 0.34 minutes, and the average half-peak width (HWHM) of the two peaks was 0.4 minutes. Based on the resolution calculation results, the first gradient segment was adjusted to 0-8 minutes, phase B 10-30%, reducing the gradient change rate by approximately 40%. After re-exercising the adjusted program, the resolution between glutamic acid and aspartic acid derivatives improved to 1.42, meeting the analytical requirements. Throughout the chromatographic separation process, a total of 20 amino acid derivatives were separated, with retention times ranging from 3.2 to 18.5 minutes. The resolution of each peak was greater than 1.2, and the peak shape showed good symmetry.

[0112] like Figure 3 As shown, Figure 3 This diagram illustrates the comparison of chromatographic separation effects before and after adjusting the gradient elution program in this embodiment. It shows the significant difference in the separation effect of key amino acid derivative pairs (aspartic acid (Asp) and glutamic acid (Glu)) before and after dynamically adjusting the gradient elution program using this technical solution. The dashed lines combined with diamond-shaped data points represent chromatograms obtained using the unoptimized initial fixed gradient program (0-5 minutes, B phase 10-30%). The retention times of the Asp and Glu peaks are approximately 4.3 minutes and 4.64 minutes, respectively, with a calculated retention time difference ΔtR of 0.34 minutes. The average half-peak width of the two peaks is 0.4 minutes, resulting in a resolution Rs of only 0.85. This value is less than 1.0, indicating severe overlap between the two peaks, which cannot meet the requirements for accurate quantitative analysis. To address this problem, this technical solution triggers a gradient adjustment mechanism based on the resolution value: since Rs < 1.0, the rate of change of the B phase in the first gradient segment is automatically reduced by approximately 40%, adjusting the B phase from 10% to 30% within 0-8 minutes. The solid lines combined with circular data points in the diagram show the chromatograms after executing the adjusted program. After adjustment, the retention times of Asp and Glu were extended to approximately 5.5 minutes and 7.5 minutes, respectively, and the baselines between the two peaks were completely separated, increasing the calculated resolution Rs to 1.42. This result meets the analytical requirement of a resolution greater than 1.2, demonstrating that this technique, by calculating the resolution in real time and dynamically adjusting the gradient rate, can effectively solve the problem of difficult separation of complex components.

[0113] The above-described chromatographic separation techniques achieved efficient separation of amino acid derivatives. Based on the polar characteristics of amino acid derivatives, an intelligent gradient optimization strategy was employed to address the challenge of traditional fixed gradient programs failing to meet the analytical needs of complex samples. The method of polarity range division and gradient program matching improved separation specificity, ensuring effective separation of various amino acid derivatives. Real-time resolution monitoring and gradient adjustment techniques dynamically optimized separation conditions to adapt to the separation requirements of different samples, enhancing the applicability and robustness of the method.

[0114] Based on the elution time window of the amino acid derivative components, the ionization polarity mode of the ion source is switched during mass spectrometry detection to ensure that the amino acid derivative components are detected in the mode with optimal ionization efficiency. The obtained mass spectrometry response signal includes:

[0115] The ultraviolet detection signal and ion current signal of the amino acid derivative component are collected. The elution time segment of the amino acid derivative component is determined by the peak position of the ultraviolet detection signal and ion current signal, and the elution time segment is set as the elution time window.

[0116] The amino acid derivative components are detected in positive and negative ionization modes according to the elution time window. Mass spectrometry signals are obtained through the positive and negative ionization modes. The signal-to-noise ratio and peak shape ratio of the mass spectrometry signals are calculated. The optimal ionization efficiency mode is determined based on the signal-to-noise ratio and peak shape ratio.

[0117] The optimal ionization efficiency mode is combined with the elution time window to generate an ionization polarity switching command, which is used to control the ionization polarity mode of the ion source during mass spectrometry detection.

[0118] The response signal output by the mass spectrometer detector is monitored, and when the response signal indicates that the amino acid derivative component has been detected, the mass spectrometer response signal of the amino acid derivative component is acquired.

[0119] The UV detection signals of amino acid derivative components were acquired using a diode array detector, with a dominant wavelength of 338 nm, a reference wavelength of 450 nm, a sampling frequency of 10 Hz, and a data acquisition range of 0-25 minutes. Ion current signals were acquired using full-scan mode, with a sampling frequency of 5 Hz and a collision energy of 10 eV. The UV detector and mass spectrometer were connected via a signal trigger circuit to ensure data acquisition time synchronization. After signal acquisition, the UV detection data were processed using a peak detection algorithm to extract the start time, peak time, and end time of each chromatographic peak.

[0120] The peak detection algorithm sets a threshold based on the rate of change of signal intensity. When the rate of change of signal intensity exceeds three times the standard deviation of background noise, it is marked as the peak start point; when the signal intensity reaches a local maximum, it is marked as the peak peak; and when the signal intensity decreases to the level of background noise, it is marked as the peak end point.

[0121] The peaks in the corresponding ion current signals are also labeled using the same principle. The UV detection signal and the ion current signal are aligned on the time axis to compensate for the transmission delay between the two detectors, which is typically 0.15 minutes. By comparing the aligned UV detection signal and the ion current signal, the elution time range for each amino acid derivative component is determined, i.e., the time range from the peak start point to the peak end point. Considering that fluctuations in chromatographic conditions may cause slight drift in retention time, each elution time range is extended by 0.1 minutes before and after, forming the final elution time window.

[0122] The amino acid derivative components within the defined elution time window were detected using both positive and negative ionization modes. In positive ionization mode, the capillary voltage of the electrospray ionization source was set to 4000 V, the desolvation gas temperature to 350 °C, the gas flow rate to 8 L / min, the nebulizer gas pressure to 40 psi, and the sheath gas flow rate to 2 L / min. In negative ionization mode, the capillary voltage was set to 3500 V, with other parameters remaining the same as in positive ionization mode.

[0123] Within each elution time window, positive and negative ionization modes are acquired alternately, with each mode acquiring for 1 second, completing at least 5 cycles of alternating acquisition. The obtained mass spectrometry signals are processed using a peak detection algorithm to calculate the signal-to-noise ratio (SNR) and peak shape ratio. The SNR is calculated as the ratio of peak signal intensity to the standard deviation of background noise; the peak shape ratio is calculated as the ratio of peak height to full width at half maximum (FWHM), reflecting the sharpness of the peak.

[0124] A higher signal-to-noise ratio (SNR) indicates higher detection sensitivity; a higher peak shape ratio indicates a sharper peak and higher quantitative accuracy. The optimal ionization efficiency mode is determined by comparing the SNR and peak shape ratios under positive and negative ionization modes. If the SNR of the positive ionization mode is more than 20% higher than that of the negative ionization mode, and the peak shape ratio is comparable or higher, then the positive ionization mode is selected as the optimal mode. If the SNR of the negative ionization mode is more than 20% higher than that of the positive ionization mode, and the peak shape ratio is comparable or higher, then the negative ionization mode is selected as the optimal mode. If the SNR differences between the two modes are less than 20%, then the mode with the higher peak shape ratio is selected as the optimal mode.

[0125] The determined optimal ionization efficiency mode is combined with the corresponding elution time window to generate an ionization polarity switching instruction table. The ionization polarity switching instruction table contains multiple time period settings, each time period corresponding to the elution time window and corresponding ionization polarity mode of an amino acid derivative component.

[0126] The ionization polarity switching command table is transmitted to the mass spectrometer control system via a time program interface, enabling automatic polarity switching during sample analysis. The time program interface uses a binary format and includes three fields: time point identifier, polarity mode identifier, and checksum, with a transmission rate of 10 times / second. Based on a comparison between the current time and the time period in the ionization polarity switching command table, the ion source's ionization polarity mode is automatically switched. The polarity mode switching time is less than 0.1 seconds, ensuring that target components are not missed. To prevent signal interference during polarity switching, a 0.2-second equilibration time is set after each switch, allowing the ion source parameters to stabilize before data acquisition begins.

[0127] The response signal output by the mass spectrometer detector is monitored in real time by the data acquisition system. A threshold trigger mechanism is used for response signal monitoring: a valid signal is marked as starting when the signal intensity exceeds five times the standard deviation of the background noise; a valid signal is marked as ending when the signal intensity remains below twice the standard deviation of the background noise for 30 consecutive data points. The monitoring time for each amino acid derivative component is no less than 1.5 times its elution time window to ensure complete acquisition of the mass spectrometry response signal of the target component.

[0128] When the mass spectrometry response signal of the target component completes its rise and fall, and the signal intensity returns to the background level, the component is considered to have been successfully detected. The criteria for successful detection also include the completion of signal integration area calculation and peak shape parameter extraction. For each successfully detected amino acid derivative component, its characteristic parameters of the mass spectrometry response signal, including peak area, peak height, retention time, signal-to-noise ratio, and mass-to-charge ratio, are recorded for subsequent qualitative and quantitative analysis.

[0129] Taking glutamate derivatives as an example, the elution time window was determined to be 5.25–5.75 minutes. Within this time window, detection was performed in both positive and negative ionization modes. The obtained mass spectrometry signals showed a signal-to-noise ratio (SNR) of 68.5 and a peak shape ratio of 4.2 in positive ionization mode, and a SNR of 32.1 and a peak shape ratio of 3.8 in negative ionization mode. Comparing the detection performance of the two modes, the positive ionization mode showed a higher SNR and a higher peak shape ratio than the negative ionization mode. Therefore, the positive ionization mode was determined to be the optimal ionization efficiency mode for glutamate derivatives. This result was combined with the elution time window to generate an ionization polarity switching command: 5.25–5.75 minutes, using positive ionization mode. The mass spectrometer automatically switched to positive ionization mode at 5.25 minutes to detect the glutamate derivative until 5.75 minutes when it switched to the optimal ionization mode for the next component. The mass spectrometry response signal of the glutamate derivative was monitored, showing that the peak started at 5.32 minutes, peaked at 5.48 minutes, and ended at 5.62 minutes. The response signal completely recorded the mass spectrometry characteristics of the glutamate derivative, including the quasi-molecular ion peak and characteristic fragment peaks, proving that the detection process was successfully completed.

[0130] This implementation overcomes the limitations of traditional fixed ionization modes, which cannot meet the detection requirements of amino acid derivatives with different polarities. It provides the most suitable ionization environment for each component, maximizing its response signal. Through the synergistic cooperation of UV detection and mass spectrometry, it achieves precise tracking of chromatographic elution behavior and intelligent switching of ionization modes, avoiding the inconvenience and delay of manual operation. Real-time automatic switching of ionization polarity improves the targeting and efficiency of data acquisition, reduces unnecessary signal interference and data redundancy, and optimizes the working state of the mass spectrometer.

[0131] Substituting the mass spectrometry response signal into a preset standard curve, the amino acid concentration values ​​corresponding to the amino acid derivative components in the sample to be tested are calculated, including:

[0132] The response intensity and response deviation of the amino acid derivative component in the mass spectrometry response signal are collected at multiple mass-to-charge ratios. The quantitative mass-to-charge ratio is selected according to the ratio of the response intensity to the response deviation value, and the response signal at the quantitative mass-to-charge ratio is obtained.

[0133] Standard response signals of amino acid derivative standard solutions of known concentrations at the given quantitative mass-to-charge ratio are collected, and a standard curve is established between the standard response signal and the concentration. The response signal is then normalized using the standard curve to obtain the standardized response value.

[0134] Substitute the standardized response value into the standard curve, calculate the conversion coefficient between the standardized response value and the concentration, and calculate the amino acid concentration value using the conversion coefficient.

[0135] Verify the accuracy of the amino acid concentration value. When the accuracy meets the measurement requirements, obtain the amino acid concentration value corresponding to the amino acid derivative component in the sample to be tested.

[0136] Mass spectrometry response signal acquisition employed multi-reaction monitoring (MRM), with three characteristic mass-to-charge ratio (MTBR) channels monitored for each amino acid derivative. The selection of the MMBR was based on the fragmentation behavior of the amino acid derivative molecules, typically including quasi-molecular ions, major fragment ions, and characteristic neutral loss ions. The mass spectrometer parameters were set as follows: residence time 50 ms, scan range ±0.5 μm, collision energy 10–30 eV, declustering voltage 5 V, spray pressure 35 psi, desolvation gas flow rate 10 L / min, and temperature 350 °C. Peak integration was performed on the acquired mass spectrometry response signals to obtain the peak area data for each MMBR, which was used as the response intensity value. Simultaneously, the corresponding response deviation value, i.e., the relative standard deviation of three consecutive repeated measurements, was calculated. The ratio of the response intensity to the response deviation value was used to evaluate the signal stability and reliability of different MMBR channels. A higher ratio indicates better signal quality for that MMBR channel. Based on the calculation results, the MMBR channel with the highest ratio was selected as the quantitative MMBR.

[0137] For lysine derivatives, the three characteristic mass-to-charge ratios were 329.2, 84.1, and 147.2, with corresponding response intensities of 68542, 124356, and 32541, and response deviations of 3.2%, 1.8%, and 5.6%, respectively. The calculated ratios were 2142, 6909, and 581. Based on this, 84.1 was selected as the quantitative mass-to-charge ratio for lysine derivatives, and the response signal at this ratio was obtained for subsequent quantitative analysis.

[0138] Standard solutions of amino acid derivatives at known concentrations were prepared using a serial dilution method, including seven concentration points covering a concentration range of two orders of magnitude. Taking lysine as an example, standard solutions with concentrations of 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 μg / mL were prepared. The standard solutions were treated under the same derivatization and chromatographic separation conditions as the analyte samples, and the standard response signals were acquired at the selected quantitative mass-to-charge ratio. Each standard concentration point was measured three times, and the average value was taken as the standard response value for that concentration point.

[0139] The standard curve was established using a weighted linear regression method, with the weighting coefficients set to the reciprocal of the concentration to ensure fitting accuracy in the low-concentration region. The standard curve equation represents the linear relationship between peak area and concentration, with the slope representing the sensitivity factor and the intercept representing the background response. The standard curve was evaluated to ensure a correlation coefficient of no less than 0.995 and a regression residual of no more than 15% at each point. After establishing the standard curve, the response signal of the sample was normalized to obtain a standardized response value, which represents the relative position of the sample's response signal within the range of the standard curve.

[0140] Amino acid concentrations were determined by mapping standardized response values ​​to a pre-established quantitative model. To reduce the impact of sample pretreatment and matrix differences on the detection results, dilution correction, derivatization efficiency correction, and matrix effect correction were introduced into the quantitative model to uniformly correct the standardized response values. Specifically, dilution correction was determined based on the volume changes experienced by the sample during pretreatment; derivatization efficiency correction was determined based on the recovery of the internal standard compound during the derivatization reaction; and matrix effect correction was determined based on the response relationship between the matrix-matched calibration curve and the standard solution calibration curve.

[0141] The dilution correction, derivatization efficiency correction, and matrix effect correction together constitute the comprehensive conversion parameters used for response correction. These comprehensive conversion parameters are used to map the standardized response value to the corresponding amino acid concentration result, thereby obtaining the quantitative output after preprocessing and matrix effect correction.

[0142] For the determination of lysine in serum samples, when the standardized response value is 8.65, the dilution factor is 10, the derivatization efficiency correction factor is 1.05, and the matrix effect correction factor is 0.92, the calculated conversion coefficient is 9.66, and the final amino acid concentration is 8.65 × 9.66 = 83.56 μg / mL.

[0143] The accuracy of amino acid concentration values ​​was verified using the spiked recovery method and the quality control sample method. The spiked recovery method involves adding a known amount of standard to the test sample and comparing the ratio of the concentration difference before and after addition to the theoretical addition amount; the recovery rate should be within the range of 85%-115%. The quality control sample method uses quality control samples of known concentrations for determination, comparing the measured results with the theoretical values; the relative error should be within ±10%. Accuracy verification also includes precision assessment, which involves calculating the relative standard deviation through six consecutive repeated determinations; the relative standard deviation should not exceed 10%.

[0144] The accuracy criteria for the amino acid concentration values ​​corresponding to the amino acid derivative components in the test sample are as follows: recovery rate within the range of 90%-110%, relative error not exceeding ±8%, and relative standard deviation not exceeding 8%. When the accuracy verification results meet the above requirements, the obtained amino acid concentration values ​​are confirmed as reliable and output as the final determination results. For the determination of lysine concentration in the aforementioned serum sample, the spiked recovery rate was 94.8%, the relative error was -3.2%, and the relative standard deviation was 4.7%, all of which met the accuracy requirements. Therefore, 83.56 μg / mL was confirmed as the final concentration value of lysine in this sample.

[0145] This invention, based on multiple reaction monitoring (MRM) and internal standard correction, significantly improves the accuracy and sensitivity of amino acid quantification analysis. A multi-mass-to-charge ratio (MTR) channel screening strategy ensures the selection of the optimal MTR for quantification, effectively avoiding the impact of co-elution interference and background noise. The weighted regression design of the standard curve balances accuracy across high and low concentration ranges, broadening the method's linear range. The introduction of normalization and conversion coefficients fully considers various influencing factors during sample pretreatment, improving the reliability of the quantification results. A multi-level accuracy verification mechanism ensures the authenticity of the measurement results, meeting the needs of trace amino acid analysis in complex biological samples.

[0146] One technical solution provided in this embodiment of the invention is an electronic device, including: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the steps in any of the aforementioned methods.

[0147] One technical solution provided in this embodiment of the invention is a computer-readable storage medium storing a computer program, wherein the processor executes the computer program to implement the steps in any of the aforementioned methods.

[0148] The specific embodiments described above are preferred embodiments of the present invention and are not intended to limit the specific scope of the present invention. The scope of the present invention includes, but is not limited to, these specific embodiments. All equivalent changes made in accordance with the shape and structure of the present invention are within the protection scope of the present invention.

Claims

1. An online pre-column derivatization coupled liquid chromatography-mass spectrometry method for amino acid detection, characterized in that, Includes the following steps: The sample to be tested was obtained and subjected to protein hydrolysis to obtain a hydrolysate containing free amino acids; After the hydrolysate is mixed with the derivatizing reagent, it is sequentially passed through multiple reaction zones arranged in series for online derivatization. Each reaction zone maintains a different pH environment for amino acids with different dissociation constants, thus obtaining a derivatization reaction solution containing amino acid derivatives. An online detection unit is set at the outlet of the multi-stage reaction zone to monitor the concentration of amino acid derivatives generated. The supply rate of the derivatizing reagent and the temperature of the reaction zone are adjusted in real time according to the concentration to obtain a mobile phase containing amino acid derivatives. The mobile phase containing amino acid derivatives was subjected to chromatographic separation to obtain amino acid derivative components separated in order of elution time. Based on the elution time window of the amino acid derivative components, the ionization polarity mode of the ion source is switched during the mass spectrometry detection process so that the amino acid derivative components are detected in the mode with the optimal ionization efficiency, and the mass spectrometry response signal is obtained. Substitute the mass spectrometry response signal into the preset standard curve to calculate the amino acid concentration value corresponding to the amino acid derivative component in the sample to be tested. The hydrolysate and derivatizing reagent are mixed and then sequentially passed through multiple reaction zones arranged in series for online derivatization. Each reaction zone maintains a different pH environment for amino acids with different dissociation constants, resulting in a derivatization reaction solution containing amino acid derivatives, including: The pH value in the hydrolysate was measured using an electrochemical detector, and the conductivity of the hydrolysate was measured using a conductivity detector. Based on the pH value and conductivity, an acid-base characteristic curve of the hydrolysate was generated. The free amino acids in the hydrolysate are classified according to the acid-base characteristic curves, the pH environment required for the free amino acids in the multi-stage reaction zone set in series is determined, and the amount of hydrolysate and derivatizing reagent is calculated. The hydrolysate and the derivatizing reagent are delivered to the eddy mixing device according to the specified dosage. The eddy mixing device uses eddy shear force to fully mix the hydrolysate and the derivatizing reagent to form a mixed reaction solution. The buffer solution is delivered to multiple reaction zones arranged in series, so that the multiple reaction zones form a pH environment adapted to different free amino acids; The mixed reaction solution is subjected to an online derivatization reaction through multiple reaction zones arranged in series. The pH changes of the multiple reaction zones are monitored in real time using a detector. The amount of buffer solution replenished is adjusted according to the pH changes to maintain the pH environment of the multiple reaction zones, thereby obtaining a derivatization reaction solution containing amino acid derivatives.

2. The method according to claim 1, characterized in that, The sample to be tested was obtained and subjected to protein hydrolysis to obtain a hydrolysate containing free amino acids, including: The sample to be tested is subjected to chemical hydrolysis and enzymatic hydrolysis in sequence. The chemical hydrolysis and enzymatic hydrolysis are carried out in a hydrolysis unit equipped with an ion exchange membrane group, which is used to remove interfering ions generated during the hydrolysis process. The pH value and conductivity of the hydrolysis products during the hydrolysis process are detected, and the degree of hydrolysis is determined based on the pH value and conductivity. Adjust the hydrolysis treatment conditions based on the degree of hydrolysis until the pH value and conductivity of the hydrolysis products reach the preset range; The hydrolysis product is subjected to ion exchange treatment through the ion exchange membrane assembly to obtain a hydrolysate containing free amino acids.

3. The method according to claim 1, characterized in that, An online detection unit is installed at the outlet of the multi-stage reaction zone to monitor the concentration of amino acid derivatives generated. Based on the generated concentration, the derivatizing reagent supply rate and the reaction zone temperature are adjusted in real time to obtain a mobile phase containing amino acid derivatives, comprising: The fluorescence signal of amino acid derivatives at the outlet of the multi-stage reaction zone is detected, and the temperature and pressure data of the multi-stage reaction zone are recorded. The concentration of amino acid derivatives generated is calculated based on the fluorescence signal, temperature data and pressure data. The formation pattern of amino acid derivatives is determined based on the correspondence between the formation concentrations under different temperature and pressure data, and the degree of completion of the derivatization reaction is calculated based on the formation pattern. The supply rate of the derivatizing reagent and the temperature of the reaction zone are adjusted by calculating the completion rate, thereby controlling the delivery speed of the derivatizing reagent and the heating power of the reaction zone. The concentration of amino acid derivatives after adjustment is monitored. When the adjusted concentration meets the chromatographic separation conditions, a mobile phase containing amino acid derivatives is obtained. The mobile phase contains the amino acid derivatives that have completed the derivatization reaction and the mobile phase solvent.

4. The method according to claim 1, characterized in that, The mobile phase containing amino acid derivatives was subjected to chromatographic separation to obtain amino acid derivative components separated in order of elution time, including: The polarity value of amino acid derivatives in the mobile phase is detected, and the polarity range of amino acid derivatives is divided according to the polarity value. Based on the polarity range, the range of change of the mobile phase gradient and the stationary phase material of the chromatographic column are determined, and a gradient elution program is generated. The gradient elution program is executed, the ultraviolet detection signal at the column outlet is collected, and the retention time difference and peak width ratio of adjacent amino acid derivatives are calculated. The resolution values ​​of adjacent chromatographic peaks are calculated based on the retention time difference and peak width ratio, and the rate of change of the gradient elution program is adjusted based on the resolution values. The adjusted gradient elution program is executed, and the ultraviolet detection signal at the column outlet is monitored. When the ultraviolet detection signal indicates that the chromatographic peak separation is complete, the amino acid derivative components separated in the order of elution time are obtained.

5. The method according to claim 1, characterized in that, Based on the elution time window of the amino acid derivative components, the ionization polarity mode of the ion source is switched during mass spectrometry detection to ensure that the amino acid derivative components are detected in the mode with optimal ionization efficiency. The obtained mass spectrometry response signal includes: The ultraviolet detection signal and ion current signal of the amino acid derivative component are collected. The elution time segment of the amino acid derivative component is determined by the peak position of the ultraviolet detection signal and ion current signal, and the elution time segment is set as the elution time window. The amino acid derivative components are detected in positive and negative ionization modes according to the elution time window. Mass spectrometry signals are obtained through the positive and negative ionization modes. The signal-to-noise ratio and peak shape ratio of the mass spectrometry signals are calculated. The optimal ionization efficiency mode is determined based on the signal-to-noise ratio and peak shape ratio. The optimal ionization efficiency mode is combined with the elution time window to generate an ionization polarity switching command, which is used to control the ionization polarity mode of the ion source during mass spectrometry detection. The response signal output by the mass spectrometer detector is monitored, and when the response signal indicates that the amino acid derivative component has been detected, the mass spectrometer response signal of the amino acid derivative component is acquired.

6. The method according to claim 1, characterized in that, Substituting the mass spectrometry response signal into a preset standard curve, the amino acid concentration values ​​corresponding to the amino acid derivative components in the sample to be tested are calculated, including: The response intensity and response deviation of the amino acid derivative component in the mass spectrometry response signal are collected at multiple mass-to-charge ratios. The quantitative mass-to-charge ratio is selected according to the ratio of the response intensity to the response deviation value, and the response signal at the quantitative mass-to-charge ratio is obtained. Standard response signals of amino acid derivative standard solutions of known concentrations at the given quantitative mass-to-charge ratio are collected, and a standard curve is established between the standard response signal and the concentration. The response signal is then normalized using the standard curve to obtain the standardized response value. Substitute the standardized response value into the standard curve, calculate the conversion coefficient between the standardized response value and the concentration, and calculate the amino acid concentration value using the conversion coefficient. Verify the accuracy of the amino acid concentration value. When the accuracy meets the measurement requirements, obtain the amino acid concentration value corresponding to the amino acid derivative component in the sample to be tested.

7. An electronic device, characterized in that, include: A memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor, when executing the computer program, implements the steps of the method as described in any one of claims 1 to 6.

8. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores computer program instructions that, when executed by a processor, implement the steps of the method as described in any one of claims 1 to 6.

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