Full-automatic online extraction-post-column derivatization combined device for aldehydes and application

Abstract

The invention discloses a full-automatic online extraction-post-column derivatization combined device for aldehydes and application of the full-automatic online extraction-post-column derivatization combined device. The device integrates on-line in-tube solid-phase microextraction, liquid chromatographic separation, post-column derivatization reaction and mass spectrometry detection modules, and realizes on-line rapid reaction of chromatographic eluent and a derivatization reagent by using a three-way mixer. The method is mainly used for full-automatic accurate analysis of endogenous aldehyde metabolic markers such as hexanal and heptanal in body fluid such as human urine and plasma, biological matrix interference is effectively removed through on-line enrichment, and the detection sensitivity is remarkably improved; meanwhile, the device can be extensively applied to rapid screening and quantification of trace aldehydes in complex samples such as traditional Chinese medicines, foods, environments and plants. The method provided by the invention realizes automation of the whole process from sample pretreatment to detection, solves the problems of tedious and time-consuming traditional off-line derivation operation and poor reproducibility, and has the advantages of high flux, low cost, no pollution to mass spectrum and the like.

Description

A fully automated online extraction-post-column derivatization coupled device for aldehydes and its application Technical Field

[0001] This invention relates to the fields of analytical chemistry and instrumentation, specifically to a fully automated online extraction-post-column derivatization device for aldehydes and its application in the analysis of aldehyde metabolites in complex samples such as body fluids, food, and traditional Chinese medicine. Background Technology

[0002] Aldehydes are widely found in the environment, food, and organisms. In the biomedical field, endogenous aldehydes (such as hexanal and heptanal) are products of lipid peroxidation and have been proven to be potential biomarkers for various diseases, including lung cancer. In the food and traditional Chinese medicine fields, aldehydes (such as cinnamaldehyde and citral) are important active ingredients or flavor compounds. Therefore, establishing rapid and sensitive methods for aldehyde detection is of great significance.

[0003] Currently, the main methods for detecting aldehydes include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Due to the high polarity, low volatility, or thermal instability of many aldehyde compounds, LC-MS has gradually become the mainstream method. However, aldehydes have extremely low ionization efficiency in electrospray ionization (ESI) sources, making direct detection difficult; therefore, chemical derivatization is necessary to introduce easily ionizable groups.

[0004] Existing derivatization methods mostly employ pre-column derivatization, meaning the sample and derivatizing reagent are manually mixed and reacted before analysis. This method has significant drawbacks:

[0005] (1) Cumbersome and time-consuming operation: It requires offline incubation, extraction, centrifugation and other steps, making it difficult to achieve high-throughput analysis;

[0006] (2) Severe matrix interference: Direct reaction with complex biological samples (such as urine) will introduce a large number of impurities;

[0007] (3) Poor reproducibility: Manual operation is prone to introducing errors.

[0008] While some studies have attempted post-column derivatization (PCD), traditional PCD devices typically require long reaction coils and heating elements to ensure complete reaction, leading to severe peak broadening and reduced separation efficiency. Furthermore, traditional in-tube solid-phase microextraction (In-tube SPME) is difficult to seamlessly integrate with post-column derivatization systems for fully automated analysis.

[0009] Therefore, there is an urgent need to develop a fully automated analytical system that integrates online pretreatment, chromatographic separation, and post-column derivatization to solve the above problems. Summary of the Invention

[0010] The purpose of this invention is to provide a fully automated online extraction-post-column derivatization coupled device for aldehydes and its application. By coupling in-tube solid-phase microextraction with post-column derivatization technology online, fully automated and highly sensitive detection of aldehydes in complex matrices is achieved.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: a fully automated online extraction-post-column derivatization coupled device for aldehydes, comprising an automatic sample injection system, a high-pressure switching valve, an in-tube solid-phase microextraction column, a liquid chromatography separation system, a post-column derivatization reaction system, and a detector. The in-tube solid-phase microextraction column is connected to the loop position of the high-pressure switching valve for online enrichment of aldehydes and removal of the matrix; the post-column derivatization reaction system mixes the chromatographic eluent and derivatizing reagent online through a three-way mixing element, and the mixture undergoes a rapid derivatization reaction in the transmission pipeline and ion source before entering the detector.

[0012] The present invention also provides a method for detecting aldehydes using the above-mentioned device, including online extraction, desorption separation, post-column derivatization and mass spectrometry detection steps.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) Fully automated: It realizes the full automation of the process from sample injection to result output without human intervention, which significantly improves analytical throughput and reproducibility.

[0015] (2) High sensitivity and anti-interference: In-tube solid-phase microextraction effectively enriches trace target substances and removes matrix interference; post-column derivatization significantly enhances the mass spectrometry response of aldehydes (detection limit down to the nM level).

[0016] (3) High chromatographic efficiency: The microdroplet reaction acceleration mechanism in the electrospray ionization process is utilized, eliminating the need for additional long reaction coils or heating devices, thus avoiding peak broadening caused by dead volume after column.

[0017] (4) High versatility: By changing the derivatizing reagent (hydroxylamine hydrochloride or ammonium sulfite) and detection mode (positive / negative ion), it can flexibly adapt to the detection needs of different matrices (urine, traditional Chinese medicine, etc.). Attached Figure Description

[0018] Figure 1 is a schematic diagram of the fully automated online extraction-post-column derivatization device for aldehydes provided in an embodiment of the present invention;

[0019] Figure 2 is a connection diagram of the post-column derivatization reaction system (three-way mixing mode) in an embodiment of the present invention;

[0020] Figure 3 is a chromatogram of hexanal and heptanal in human urine detected using the device of the present invention (corresponding to Example 2);

[0021] Figure 4 is a chromatogram of trans-cinnamaldehyde in cinnamon extract detected using the device of the present invention in negative ion mode (corresponding to Example 3). Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1: Construction of a Fully Automated Detection Device

[0024] As shown in Figure 1, the device constructed in this embodiment mainly includes:

[0025] (1) Liquid chromatography system: The Shimadzu LC-20AD system was used, equipped with three pumps (pumps A and B for chromatographic gradient elution, and pump C for in-tube extraction and loading), an autosampler (SIL-20A) and a column oven.

[0026] (2) High pressure switching valve: A six-way valve is used to switch between the "Load" and "Inject" states.

[0027] (3) In-tube solid-phase microextraction column (In-tube SPME): A self-made poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic column (Poly(MAA-co-EDMA)) with a length of about 4cm is installed at the metering ring position of the six-way valve for adsorbing aldehydes.

[0028] (4) Analytical column: Inertsil ODS-3 column (250mm×2.1mm, 5μm).

[0029] (5) Post-column derivatization module: Derivatization reagents are delivered using a precision syringe pump (Harvard Apparatus) and connected to the column outlet via a zero dead volume tee. The tee outlet is connected to the mass spectrometer via a section of PEEK tubing (10-80 cm long, 100 μm inner diameter).

[0030] (6) Detector: Triple quadrupole tandem mass spectrometer (AB Sciex QTRAP 3200 or Shimadzu MS-8040).

[0031] Example 2: Detection of lung cancer markers (hexanal, heptanal) in human urine (Group A reagent method)

[0032] This embodiment utilizes the apparatus described in Example 1 to establish a fully automated detection method for lung cancer biomarkers (hexanal and heptanal) in human urine. Hydroxylamine hydrochloride (HAHC) is used as the derivatizing reagent, and the detection mode is positive ion mode.

[0033] The specific steps are as follows:

[0034] (1) Sample pretreatment: Collect human urine samples and centrifuge at 14,000 rpm for 15 minutes at 4 degrees Celsius. Take the supernatant, dilute it 3 times with phosphate buffer (25 mM, pH 3.5), and place it in an autosampler bottle for testing.

[0035] (2) Online extraction process: Switch the six-way valve to the "Load" position. The autosampler draws 800 μL of sample and injects it into the system. Pump C delivers washing solution (25 mM phosphate buffer) at a flow rate of 80 μL / min, loading the sample into the solid-phase microextraction column. During this process, the target aldehydes are adsorbed onto the entire column, while matrix interfering substances such as proteins and salts in the urine are directly discharged into the waste container by the washing solution.

[0036] (3) Desorption and Separation Process: After extraction, switch the six-way valve to the "Inject" position. Pumps A and B start the gradient elution program. The mobile phase (methanol / water, 65:35, v / v) flows through the solid-phase microextraction column in the tube, eluting the enriched aldehydes in reverse and carrying them into the analytical chromatographic column (Inertsil ODS-3, 250 mm x 2.1 mm, 5 μm) for separation. The chromatographic flow rate is set to 0.2 mL / min, and the column temperature is 40 degrees Celsius.

[0037] (4) Post-column derivatization and detection: A precision syringe pump continuously delivered the derivatizing reagent at a flow rate of 2 μL / min. The derivatizing reagent was a 20 mM hydroxylamine hydrochloride solution (solvent: 50% methanol-water). The column eluent and derivatizing reagent were mixed at the tee and then introduced into the mass spectrometer. The mass spectrometer was set to positive ion multiple reaction monitoring (MRM) mode, and the ion source temperature was set to 400°C. Hexanal and heptanal reacted with hydroxylamine hydrochloride to generate the corresponding oxime derivatives, which were then detected.

[0038] Experimental results show that this method can effectively remove urine matrix interference, with detection limits (LODs) of 15 nM and 9 nM for hexanal and heptanal, respectively. The relative recoveries ranged from 95.4% to 116.8%, and the intra-day and inter-day relative standard deviations (RSDs) were both less than 17.2%. The entire process requires no manual intervention, and the analysis cycle for a single sample is approximately 35 minutes.

[0039] Example 3: Screening of aldehydes in traditional Chinese medicine and plants based on ammonium sulfite derivatization method

[0040] This embodiment utilizes the apparatus described in Example 1 to establish a rapid screening and quantification method for aldehydes in complex plant matrices (such as cinnamon and lemongrass). Ammonium sulfite was used as the derivatizing reagent, and the detection mode was negative ion mode.

[0041] The specific steps are as follows:

[0042] (1) Sample preparation: Weigh out Chinese medicine powder (such as the contents of cinnamon capsules) or plant powder (such as lemongrass), add methanol for ultrasonic extraction, centrifuge, take the supernatant, dilute with water by 1 time, and then inject the sample.

[0043] (2) Post-column derivatization conditions: The derivatizing reagent was a 100 mM ammonium sulfite aqueous solution delivered by a precision syringe pump at a flow rate of 2 μL / min. The chromatographic separation conditions were the same as in Example 2, and the mobile phase was a methanol / water system.

[0044] (3) Mass spectrometry detection: The mass spectrometer was set to negative ion scanning mode. Aldehydes undergo nucleophilic addition reactions with ammonium sulfite to generate α-hydroxyalkane sulfonates. In collision-induced dissociation (CID) mode (collision energy 1-20 eV), all aldehyde derivatives will produce the characteristic diagnostic ion HSO3- (m / z 81). Utilizing this characteristic, the precursor ion scan (PIS 81) mode was used to screen samples for unknown aldehydes.

[0045] Experimental results showed that this method successfully screened and identified trans-cinnamaldehyde and 2'-methoxycinnamaldehyde in cinnamon extract, and successfully identified citral in lemongrass extract. The method exhibits extremely high selectivity with negligible matrix effects. For quantitative analysis, the linear range for trans-cinnamaldehyde was 0.2–20 μg / mL, with a detection limit as low as 0.03 μg / mL. Furthermore, the ammonium sulfite reagent readily decomposes into a gas at high temperatures, preventing contamination of the mass spectrometer, and is inexpensive.

[0046] Example 4: Optimization and Validation of Post-Column Reaction Conditions

[0047] This embodiment verifies the key parameters of the post-column derivative system.

[0048] Traditional post-column derivatization techniques typically require long reaction coils and heating devices to ensure complete reaction, but this leads to severe peak broadening. This invention experimentally demonstrates that the microdroplet reaction acceleration effect during electrospray ionization (ESI) can eliminate the need for a heating coil.

[0049] The effect of the length of the connecting tube (PEEK tube, 100 μm inner diameter) between the outlet of the three-way mixer and the inlet of the ion source on the detection signal was investigated. The test length ranged from 10 cm to 80 cm. The results showed that when the tube length varied within the above range, the peak area of ​​aldehyde derivatives did not differ significantly, and the chromatographic peak shape remained good without obvious peak broadening.

[0050] This indicates that the derivatization reaction of the present invention mainly occurs during the transport process after mixing and the evaporation of ESI microdroplets, and the reaction rate is extremely fast. A 10cm long connecting tube is preferably used, at which point the post-column dead volume is only 0.78 μL, maximizing the preservation of chromatographic separation efficiency.

[0051] Example 5: Methodology Validation and Actual Sample Detection Data

[0052] Using the apparatus and method described in this invention, the linear range, limit of detection (LOD), precision, and spiked recovery of the method were comprehensively validated.

[0053] (1) Linear relationship and detection limit

[0054] The detection sensitivity of the target aldehydes by the hydroxylamine hydrochloride method (under the conditions of Example 2) and the ammonium sulfite method (under the conditions of Example 3) was investigated. The experimental results (see Table 1) show that the device of this invention, combining online extraction and post-column derivatization techniques, significantly reduces the detection limit. The ammonium sulfite method showed slightly better sensitivity than the hydroxylamine hydrochloride method, with detection limits for hexanal and heptanal as low as 2-3 nM. For the active ingredient trans-cinnamaldehyde from traditional Chinese medicine, the linear correlation coefficient (R0) of this method was [not specified]. 2 () greater than 0.995.

[0055] Table 1. Detection performance indicators of the device of the present invention for different aldehydes.

[0056]

[0057]

[0058] 2. Accuracy and Precision

[0059] To verify the device's anti-interference capability in complex matrices, different concentrations of standards were added to healthy human urine and traditional Chinese medicine extracts, and the spiked recovery rate and relative standard deviation were calculated. The results (see Table 2) show that even in complex biological and plant matrices, the online in-tube solid-phase microextraction module of this invention can effectively remove interference, ensuring the accuracy of the quantitative results.

[0060] Table 2. Results of spiked recovery and precision experiments in actual samples.

[0061]

[0062] 3. Chromatographic analysis of typical samples

[0063] Using the device of this invention to analyze urine samples from lung cancer patients, the resulting chromatograms (see attached diagram) showed clear peaks of hexanal and heptanal at retention times of 20.1 min and 22.5 min, respectively, with stable baselines and no interference from impurities. When screening cinnamon extract using the ammonium sulfite method, trans-cinnamaldehyde and its isomers were clearly distinguished by precursor ion scanning (PISm / z 81), demonstrating the screening capability of the device.

[0064] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications and equivalent substitutions made by those skilled in the art based on the technical solutions of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A fully automated online extraction-post-column derivatization coupled device for aldehydes, characterized in that, include: The device comprises an automated sample introduction system, a high-pressure switching valve, an in-tube solid-phase microextraction column, a liquid chromatography separation system, a post-column derivatization reaction system, and a detector. The automated sample introduction system is connected to the first interface of the high-pressure switching valve for sample delivery. The in-tube solid-phase microextraction column is connected to the loop position of the high-pressure switching valve for online enrichment and purification of aldehydes in the sample. The liquid chromatography separation system includes a chromatographic pump and a chromatographic column. The chromatographic pump is connected to the high-pressure switching valve to elute the target analytes enriched on the in-tube solid-phase microextraction column and deliver them to the chromatographic column for separation. The post-column derivatization reaction system includes a derivatization reagent delivery pump, a mixing element, and reaction tubing. The inlet of the mixing element is connected to the outlet of the chromatographic column and the outlet of the derivatization reagent delivery pump, and the outlet of the mixing element is connected to the detector. The device achieves automated operation of sample loading extraction mode and desorption injection mode by controlling the switching of the high-pressure switching valve.

2. The fully automated online extraction-post-column derivatization coupled device for aldehydes according to claim 1, characterized in that, The adsorption medium of the solid-phase microextraction column inside the tube is a polymer monolithic column; preferably, the polymer monolithic column is a polymethacrylate-ethylene glycol dimethacrylate monolithic column.

3. The fully automated online extraction-post-column derivatization coupled device for aldehydes according to claim 1, characterized in that, The mixing element is a zero dead volume tee; the derivatization reagent delivery pump is a precision injection pump used to deliver the derivatization reagent at a constant low flow rate.

4. The fully automated online extraction-post-column derivatization coupled device for aldehydes according to claim 1, characterized in that, The detector is a tandem mass spectrometer; the length of the tubing between the outlet of the mixing element and the inlet of the mass spectrometer ion source has been optimized, ranging from 10 cm to 80 cm, in order to control the reaction time and reduce chromatographic peak broadening.

5. The fully automated online extraction-post-column derivatization coupled device for aldehydes according to claim 1, characterized in that, The device is an integrated, fully automated, online system. The extraction process, chromatographic separation process, post-column derivatization process, and detection process of the solid-phase microextraction column inside the tube are automated and continuously operated through program control.

6. A method for detecting aldehydes using the apparatus according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Online extraction: The sample to be tested is injected into the solid phase microextraction column in the tube. The aldehyde target is enriched by the extraction column, and the matrix interference is removed by the washing solution. (2) Desorption and separation: The high pressure switching valve is switched, and the aldehyde target adsorbed on the extraction column is eluted to the liquid chromatography column by the mobile phase for separation. (3) Post-column derivatization: The aldehyde-containing eluent flowing out of the chromatographic column is combined with the derivatization reagent delivered by the derivatization reagent delivery pump in the mixing element. (4) Detection: The mixture enters the detector and undergoes a derivatization reaction during the transport and ionization process to generate corresponding derivative products, which are then detected.

7. The method according to claim 6, characterized in that, The sample to be tested is selected from any one of biological fluids, traditional Chinese medicine extracts, food extracts, or environmental water samples; when the sample to be tested is a biological fluid, the biological fluid includes human urine or plasma, and the aldehyde target is an endogenous metabolic marker, including hexanal and heptanal; when the sample to be tested is a traditional Chinese medicine extract, the traditional Chinese medicine includes cinnamon or lemongrass, and the aldehyde target includes trans-cinnamaldehyde, citral, or trans-2-hexenal.

8. The method according to claim 6, characterized in that, The derivatizing reagent is selected from any one of the following groups A or B: Group A: hydroxylamine hydrochloride solution; the detector adopts a positive ion scanning mode, and the detected product is an aldehyde oxime derivative; Group B: ammonium sulfite solution; the detector adopts a negative ion scanning mode, and the detected product is an aldehyde bisulfite addition derivative, and the detected characteristic ion is the bisulfite ion (m / z 81).

9. The method according to claim 8, characterized in that, When ammonium sulfite of group B is selected as the derivatization reagent, the mass spectrometry detection adopts the collision-induced dissociation mode, and the collision energy is set to 1eV to 20eV. Qualitative or quantitative analysis is performed by monitoring the characteristic diagnostic ion bisulfite ion, or precursor ion scanning is performed to screen for unknown aldehydes.

10. The method according to claim 6, characterized in that, The post-column derivatization reaction mainly occurs in the mixed transport pipeline and during the evaporation of microdroplets from electrospray ionization, without the need for additional heating of the reaction coil.

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