A deuterated chlorogenic acid homologue, preparation method and application thereof

Abstract

The application discloses a kind of deuterated chlorogenic acid heteronuclear body and preparation method thereof.Deuterated chlorogenic acid heteronuclear body is the mixture of chlorogenic acid isotopic heteronuclear body containing different number of deuterium atoms.Preparation method includes: dissolving chlorogenic acid in deuterated methanol, hydrogen-deuterium exchange reaction is carried out in the presence of anhydrous aluminum chloride in airtight container, after reaction is completed, solvent is removed, and the deuterated chlorogenic acid heteronuclear body is obtained.The application overcomes the technical problems of irreversible poisoning of ortho-benzenediol to noble metal catalyst and hydrolysis of ester bond caused by strong acid and strong base, realizes deuterium labeling with high deuterium substitution rate, high regioselectivity and high substrate recovery rate, and is simple and easy to operate and low in cost.The mass-to-charge ratio of deuterated chlorogenic acid heteronuclear body exceeds the range of 13C isotopic envelope of natural chlorogenic acid, and there is no cross interference in MRM channel, which can be directly used as stable isotope internal standard in quantitative analysis of liquid chromatography-tandem mass spectrometry, and fills the global market gap.

Description

Technical Field

[0001] This invention belongs to the field of isotope-labeled compound technology, specifically relating to a deuterated chlorogenic acid, its preparation method, and its application. Background Technology

[0002] Chlorogenic acid (3-O-caffeoylquinic acid, CA) is a hallmark active ingredient in dozens of medicinal plants and functional foods, including honeysuckle, eucommia, and coffee beans, possessing various pharmacological activities such as antioxidant, anti-inflammatory, hypoglycemic, and neuroprotective effects. The Chinese Pharmacopoeia uses CA content as a quality control indicator for many traditional Chinese medicinal materials, including honeysuckle. However, pharmacokinetic and bioequivalence studies of CA urgently require highly accurate LC-MS / MS quantitative methods, and the matrix effect during electrospray ionization (i.e., the inhibition or enhancement of analyte ionization efficiency by co-eluent matrix components) is the most significant bottleneck restricting its quantitative accuracy.

[0003] Stable isotope dilution analysis (SIDA) is widely recognized as the "gold standard" method for eliminating matrix effects in LC-MS / MS. SIDA involves adding a stable isotope-labeled analog of the analyte to the sample as an internal standard. It utilizes the highly similar chromatographic retention behavior and mass spectrometric ionization efficiency between the internal standard and the analyte to perform quantification using the analyte / internal standard response ratio, thereby eliminating matrix effects and pretreatment losses.

[0004] However, according to research, there are currently no deuterated chlorogenic acid (CA-d) standards available on the global market. The mainstream stable isotope-labeled chlorogenic acid products are Chlorogenic acid- 13 C3, priced over $200 / mg, represents a significant market gap that has become a real bottleneck restricting high-accuracy bioanalysis and quality control in CA. Summary of the Invention

[0005] This invention provides a deuterated chlorogenic acid isoform, its preparation method, and its application, in order to solve the technical problems mentioned in the background art.

[0006] The technical solution adopted in this invention includes:

[0007] A deuterated chlorogenic acid isotope, wherein the deuterated chlorogenic acid isotope is a mixture of chlorogenic acid isotope isotopes containing different numbers of deuterium atoms (defined as CA-d1, CA-d2, CA-d3, and CA-d4 respectively, based on the number of hydrogen atoms replaced by deuterium atoms in the molecule being 1 to 4), and the main components of the mixture include CA-d3 and CA-d4. 4， The CA-d3 ([M) H] - m / z356) and CA-d4 ([M H] -The mass-to-charge ratio of chlorogenic acid (m / z 357) exceeds that of natural chlorogenic acid. 13 The C isotope envelope range is defined, and there is no isotope overlap or interference between the CA-d3 and CA-d4 multiple reaction monitoring channels and the analysis channel for undeuterated chlorogenic acid.

[0008] This invention provides a class of deuterated chlorogenic acid products, aiming to fill a long-standing market gap in this field. Given the natural isotopic abundance of carbon and hydrogen in chlorogenic acid molecules, the molecular ion peaks and characteristic fragment peaks of its low-deuteration products (such as CA-d1 ​​and CA-d2) still partially overlap with the natural isotopic peak groups in mass spectrometry analysis, affecting the specificity of detection. Therefore, the development of high-deuteration products (CA-d3 and CA-d4) has significant practical implications. The deuterated chlorogenic acid isomer mixture described in this invention, with CA-d3 and CA-d4 as the main components, has a mass-to-charge ratio (m / z) that completely exceeds that of natural chlorogenic acid. 13 The envelope range generated by the C isotope distribution has no cross-response between its detection channel and potential endogenous interfering substances in multiple reaction monitoring (MRM) mode, thus it can be directly used as a stable isotope internal standard in LC-MS / MS quantitative analysis.

[0009] As a further preferred embodiment of the above technical solution, based on the normalized peak area calculation of negative ion electrospray ionization mass spectrometry, the combined peak area of ​​CA-d3 and CA-d4 accounts for ≥50% of the total chlorogenic acid-related peak area. The total deuteration rate of CA-d3 and CA-d4 in this invention is not less than 50%. At this deuteration level, the product already possesses the basic requirements for serving as a stable isotope internal standard, and its mass spectrometric behavior can effectively avoid interference from natural isotopes, thus establishing its practical value in trace quantitative detection.

[0010] As a further preferred embodiment of the above technical solution, the peak area of ​​CA-d3 and CA-d4 accounts for ≥90% of the total chlorogenic acid-related peak area, calculated by normalizing the peak area of ​​negative ion electrospray ionization mass spectrometry.

[0011] As a further preferred embodiment of the above technical solution, in the structure of the deuterated chlorogenic acid isomer, the deuterium atom is located only in the caffeoyl part of the chlorogenic acid molecule, and the quinic acid ring does not contain a deuterium atom.

[0012] Based on the same technical concept, the present invention also provides a method for preparing the deuterated chlorogenic acid isomer described in the above technical solution, comprising the following steps: dissolving chlorogenic acid in deuterated methanol, heating in a closed container in the presence of anhydrous aluminum trichloride to carry out a hydrogen-deuterium exchange reaction, removing the solvent after the reaction is completed, and obtaining the deuterated chlorogenic acid isomer.

[0013] The inventors discovered that the CA molecule is composed of a caffeoyl moiety (containing a catechol structural unit) linked to a quinic acid moiety via an ester bond. This bifunctional structure presents three challenges for its deuteration labeling: First, the ester bond is fragile. The caffeoyl-quinic acid ester bond is easily hydrolyzed and broken under both strongly acidic (pH < 1) and strongly alkaline (pH > 10) conditions. Once hydrolyzed, the CA molecule irreversibly degrades into two fragments: caffeic acid and quinic acid. Second, it is sensitive to catechol oxidation. The phenolic hydroxyl group of catechol is readily oxidized to o-benzoquinone under alkaline conditions and / or in aerobic environments, accompanied by irreversible molecular rearrangement and polymerization. Third, it is poisoned by transition metal catalysts. Catechol is a classic bidentate ligand that can form exceptionally stable five-membered ring chelates with various transition metal ions. Therefore, CA itself is a highly efficient metal catalyst poisoning agent.

[0014] Therefore, the fundamental reason why existing H / D exchange methods generally fail in CA deuteration labeling is a structural contradiction: the catechol aromatic ring is an ideal target for deuteration reactions, but catechol is also a lethal poison to catalysts, while ester bonds exclude strong acid / strong base conditions. This is the fundamental technical reason for the current lack of deuterated chlorogenic acid products on the market.

[0015] Based on this, the present invention proposes a novel catalytic pathway: selecting Lewis acid metal ions Al 3+ This allows it to form a reversible coordination activation complex with catechol, thereby transforming catechol from a catalyst poison into a driving force for catalytic activation.

[0016] In CD3OD solution, after CA was mixed with equimolar amounts of AlCl3, the characteristic absorption peak of CA red-shifted from 327 nm to 333 nm and broadened significantly, showing a similar effect to AlCl3. 3+ – The spectral characteristics of the catechol complex were consistent. After separation by HPLC with acidic mobile phase, the complex dissociated, and the UV spectrum recovered to be consistent with that of pure CA – confirming that the coordination is a reversible process. A DCl-only catalytic control experiment (10 mmol / L) showed a near-zero deuteration rate and rapid CA degradation, ruling out the substitution mechanism of AlCl3 catalyzing the Brønsted acid SEAr through the formation of DCl with CD3OD. Substrate-activity relationship (SAR) studies revealed that substrates containing only the catechol structural unit (chlorogenic acid, caffeic acid, catechol) can undergo H / D exchange; monophenols (phenol, guaiacol, 4-methoxyphenol), catechol-free arylacrylic acids (cinnamic acid), and non-aromatic hydrocarbons (quinic acid) do not undergo deuteration at all; 3-methoxyphenol (ortho-methoxy blocking bidentate chelation) yields only a limited deuteration rate. These pieces of evidence consistently support AlCl3's ability to deuterate. 3+ – Selective H / D switching mechanism guided by catechol five-membered ring chelation.

[0017] As a further preferred embodiment of the above technical solution, the heating is intermittent, that is, the reaction system is heated to 80-120°C and held for 10-20 minutes, and then the heating is stopped and the system is allowed to cool naturally for 30-60 minutes. The above operation is repeated 2-5 times.

[0018] As a further preferred embodiment of the above technical solution, the reaction system is heated to 100°C and held for 13 minutes, then the heating is stopped and the system is allowed to cool naturally for 50 minutes. This operation is repeated 3 times.

[0019] As a further preferred embodiment of the above technical solution, the concentration of anhydrous aluminum trichloride in the reaction system is 5–20 mmol / L.

[0020] As a further preferred embodiment of the above technical solution, the concentration of chlorogenic acid in deuterated methanol is 1–20 mg / mL.

[0021] As a further preferred embodiment of the above technical solution, the concentration of chlorogenic acid in the deuterium-containing substrate alcohol is 4 mg / mL.

[0022] As a further preferred embodiment of the above technical solution, the deuterium-containing substrate includes at least one of heavy water, deuterated acetonitrile, deuterated hydrochloric acid, deuterated methanol and deuterated acetone, and is further preferably anhydrous CD3OD with a deuterium atom abundance ≥99.8 atom.

[0023] As a further preferred embodiment of the above technical solution, the solvent is removed by vacuum concentration after the reaction is completed, without column chromatography or preparative high-performance liquid chromatography purification.

[0024] As a further preferred embodiment of the above technical solution, in the obtained deuterated chlorogenic acid isomer, the combined peak area of ​​CA-d3 and CA-d4 accounts for ≥92.7% of the total chlorogenic acid-related peak area, and the substrate recovery rate is ≥88.3%.

[0025] As a further preferred embodiment of the above technical solution, the relative standard deviation of the peak area ratio of CA-d3 to CA-d4 in the deuterated chlorogenic acid isomers prepared in different batches is ≤5%.

[0026] Based on the same technical concept, the present invention also provides an application of the deuterated chlorogenic acid isotope described in the above technical solution, wherein the deuterated chlorogenic acid isotope is used as a stable isotope internal standard in quantitative analysis by liquid chromatography-tandem mass spectrometry.

[0027] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a deuterated chlorogenic acid product that has been long lacking in the global market. The deuterated chlorogenic acid isomer is mainly composed of CA-d3 and CA-d4, with a total deuteration rate ≥50%, and the mass-to-charge ratio of CA-d3 and CA-d4 exceeds that of natural CA. 13 The C isotope envelope range and the MRM channel have no cross-interference, making it suitable for direct use as a stable isotope internal standard in LC-MS / MS quantitative analysis.

[0028] (2) This invention overcomes the structural contradiction of irreversible poisoning of transition metal catalysts by catechol. Using AlCl3 as a catalyst, the deuteration rate is ≥92.7% within an effective total heating time of about 39 minutes.

[0029] (3) The reaction conditions of this invention are mild, the ester bonds remain intact, and the rapid degradation of CA caused by strong acid or strong base conditions is avoided. The substrate recovery rate is ≥88.3%.

[0030] (4) This invention achieves highly regioselective deuteration, with deuterium atoms introduced only into four specific positions of the caffeoyl moiety, and the quinic acid ring does not contain deuterium atoms, ensuring a high degree of consistency between the internal standard and the fragmentation behavior of the analytical mass spectra.

[0031] (5) This invention is simple to operate and has low raw material costs. Compared with the Chlorogenic acid- which requires a total synthesis method for preparation, it is much easier to produce. 13 For C3 products, this invention only requires deuteration labeling of chlorogenic acid molecules, which greatly reduces production costs. After the reaction is completed, only reduced pressure concentration is needed to remove the solvent, without the need for column chromatography or preparative HPLC purification.

[0032] (6) The process of this invention is stable, with good batch-to-batch consistency, and is suitable for mass production. Attached Figure Description

[0033] Figure 1 The image shows the ESI full-scan mass spectrum of the negative ion of the deuterated chlorogenic acid isoform of Example 1.

[0034] Figure 2 The deuterated chlorogenic acid isoform of Example 1 1 H NMR spectrum.

[0035] Figure 3 The images show LC-MS / MS fragment spectra of the deuterated chlorogenic acid isomers CA-d3 and CA-d4 from Example 1.

[0036] Figure 4 The bar charts show the comparison of CA H / D exchange efficiency (DI%) and substrate recovery (SR%) between Example 1 and Comparative Examples 1-4.

[0037] Figure 5Line graph comparing the deuteration rate (DI%) and substrate recovery rate (SR%) of CA in Examples 1 and 7. Detailed Implementation

[0038] The technical solution of the present invention will be further explained and described below with reference to specific embodiments.

[0039] Example 1: The deuterated chlorogenic acid isoform of this embodiment was prepared using the following method: (1) Weigh 4.0 mg (11.3 μmol) of chlorogenic acid (purity ≥98%) and place it in a 5 mL stainless steel pressure vessel lined with polytetrafluoroethylene (PTFE).

[0040] (2) Add 1.0 mL of anhydrous CD3OD (deuterium atom abundance ≥99.8 atom%) and shake to completely dissolve chlorogenic acid.

[0041] (3) Add 1.3 mg of anhydrous AlCl3 (10.0 μmol) to make the concentration of AlCl3 in the reaction system 10 mmol / L.

[0042] (4) Seal the pressure vessel and place it in an oven preheated to 100°C for 13 minutes. Then remove it and let it cool naturally for 50 minutes. Repeat the above heating and cooling operation 3 times (the total effective heating time is about 39 minutes).

[0043] (5) After the reaction is complete, cool to room temperature, open the container, transfer the reaction solution to a round bottom flask, concentrate under reduced pressure at 40°C to dryness, and obtain a pale yellow solid product, which is the deuterated chlorogenic acid isomer of this embodiment.

[0044] Mass spectrometry analysis (negative ion ESI full scan MS, using Shimadzu LCMS-8050) was performed on the deuterated chlorogenic acid isomer of this embodiment. Figure 1 As shown in the full-scan mass spectrum of the negative ion ESI, the product is at m / z 356.2 (CA-d3, [MH]). - The strongest peaks are at 357.2 (CA-d4) and 357.2 (CA-d0), while the peak at 353.2 (CA-d0) is extremely weak. After peak area normalization, the combined peak area of ​​CA-d3 and CA-d4 accounts for 92.7% of the total peak area of ​​chlorogenic acid (deuteration rate, DI), while CA-d2 accounts for only 7.3%.

[0045] Using the peak area of ​​unreacted chlorogenic acid standard at the same concentration as 100%, the sum of the peak areas of all chlorogenic acid-related peaks in the product was calculated, yielding a substrate recovery (SR) of 88.3% (sum of peak areas of all CA isoforms in the product) / (peak area of ​​CA standard at the same initial concentration). Transesterification byproducts <3%. The deuterated chlorogenic acid isomer of this embodiment was analyzed by proton nuclear magnetic resonance spectroscopy (1H NMR). 1 ¹H NMR, 700 MHz, CD₃OD: Integrating with H⁻⁴ax (δ 4.23, ¹H) on the quinic acid ring as an internal standard. Figure 2 As shown, the CA-d product 1 In the HNMR spectrum, the proton signals in the caffeoyl region H-2', H-5', H-6', and H-7' were significantly weakened, while the proton signals of the quinic acid ring (δ3.8–4.3 and 2.0–2.2) were completely consistent with those of undeuterated chlorogenic acid, indicating that the quinic acid ring does not contain deuterium atoms. The deuteration rates at each position were: H-2' (δ7.05) 92%, H-5' (δ6.78) 92%, H-6' (δ6.97) 72%, H-7' (β-alkene, δ7.54) 91%, and H-8' (α-alkene, δ6.32) 0%.

[0046] LC-MS / MS fragmentation analysis was performed on the deuterated chlorogenic acid isomer of this embodiment: as follows Figure 3 As shown, the fragment ions of CA-d0 are m / z 191.1 (quinic acid) and 179.1 (caffeoyl); as Figure 3 As shown in (a), the fragment ions of CA-d3 are m / z 191.1 and 182.1 (caffeoyl-d3); as Figure 3 As shown in (b), the fragment ions of CA-d4 are m / z 191.1 and 183.1 (caffeoyl-d4). This confirms that all deuterium atoms are located in the caffeoyl moiety.

[0047] HPLC-DAD analysis of the deuterated chlorogenic acid isomer of this embodiment showed that the product and the chlorogenic acid standard were similar in color at C1. 18 The retention time on the column was consistent (tR≈8.46 min), and the UV spectrum λ max =327 nm, consistent with chlorogenic acid.

[0048] Five independent batches were prepared using the method described in this embodiment. The statistical results showed that the relative standard deviation (RSD) of the total peak area ratio of CA-d3 to CA-d4 was 3.1%.

[0049] Example 2: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1, except that the amount of chlorogenic acid weighed in step (1) is 10 mg.

[0050] Example 3: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1, except that the amount of chlorogenic acid weighed in step (1) is 20 mg.

[0051] Example 4: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1, except that the amount of chlorogenic acid weighed in step (1) is 40 mg.

[0052] Analysis of the deuterated chlorogenic acid isomers of Examples 2-4 showed that transesterification byproducts increased significantly when the substrate concentration was ≥10 mg / mL, and the substrate concentration of 4 mg / mL in Example 1 achieved the best results.

[0053] Example 5: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1, except that after adding anhydrous AlCl3 in step (3), the concentration of AlCl3 in the reaction system is 5 mmol / L.

[0054] Example 6: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1, except that after adding anhydrous AlCl3 in step (3), the concentration of AlCl3 in the reaction system is 20 mmol / L.

[0055] Example 7: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1. The only difference is in step (4). In this embodiment, a heating scheme of heating continuously at 100°C for 39 minutes and then naturally cooling is adopted.

[0056] The deuteration rate (DI%) and substrate recovery rate (SR%) of CA in Example 1 and this Example are compared, and the comparison line graph is shown below. Figure 5 As shown ( Figure 5 (a) shows the results of Example 1. Figure 5 (b) is the result of this embodiment. Figure 5 It can be seen that in Example 1, DI = 92.7% and SR = 88.3%, while in this example, DI = 73.5% and SR = 71.2%. Under the same effective heating time, the intermittent mode is significantly better than the continuous mode.

[0057] Example 8: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1. The only difference is in step (4). In this embodiment, the heating scheme of intermittent heating at 80°C for 39 minutes followed by natural cooling is adopted.

[0058] Example 9: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 7. The only difference is in step (4). In this embodiment, the heating scheme of naturally cooling after intermittent heating at 120°C for 39 minutes is adopted.

[0059] Mass spectrometry analysis was performed on the deuterated chlorogenic acid isomers of Examples 1, 8, and 9. In the product of Example 8, the peak area ratio of CA-d1 ​​and CA-d2 increased significantly, and the deuteration rate DI decreased compared with Example 1. Although the deuteration rate of the product of Example 9 increased slightly, the substrate recovery rate SR decreased significantly. Considering all factors, the temperature of Example 1 can achieve the best results.

[0060] Example 10: The preparation method of the deuterated chlorogenic acid isomer in this embodiment is basically the same as that in Example 1, except that the deuterated substrate is replaced by deuterated acetone instead of anhydrous deuterated methanol.

[0061] Although the deuterated acetone system produces deuterated products, the area of ​​impurity peaks accounts for 18.0±1.2% (n=3), which is attributed to the ketone condensation side reaction.

[0062] Comparative Example 1: The preparation method of the deuterated chlorogenic acid isomer in this comparative example is basically the same as that in Example 1, except that in step (1), AlCl3 is replaced with an equimolar Al(OTf)3.

[0063] like Figure 4 As shown, mass spectrometry analysis of the deuterated chlorogenic acid isomer of this comparative example showed that the deuteration rate (DI) of this example was 85.4%, the substrate recovery rate (SR) was 82.1%, and the strong Lewis acidity led to an increase in transesterification byproducts compared to AlCl3.

[0064] Comparative Example 2: The preparation method of the deuterated chlorogenic acid isomer in this comparative example is basically the same as that in Example 1, except that in step (1), AlCl3 is replaced with an equimolar amount of ZnCl2.

[0065] like Figure 4 As shown, mass spectrometry analysis of the deuterated chlorogenic acid isomer of this comparative example revealed that the deuteration rate (DI) of this embodiment was close to 0, and Zn 2+ Lewis acids are too weak to activate the CH bonds in aromatic rings.

[0066] Comparative Example 3: The preparation method of the deuterated chlorogenic acid isomer in this comparative example is basically the same as that in Example 1, except that in step (1), AlCl3 is replaced with an equimolar amount of Zn(OTf)2.

[0067] like Figure 4 As shown, mass spectrometry analysis of the deuterated chlorogenic acid isomer of this comparative example revealed that the deuteration rate (DI) of this embodiment was close to 0, and Zn 2+ Lewis acids are too weak to activate the CH bonds in aromatic rings.

[0068] Comparative Example 4: The preparation method of the deuterated chlorogenic acid isomer in this comparative example is basically the same as that in Example 1, except that AlCl3 is not added in step (1).

[0069] like Figure 4 As shown, mass spectrometry analysis of the deuterated chlorogenic acid isomer of this comparative example showed that the deuteration rate (DI) of this example was 0.

[0070] Comparative Example 5: The deuterated chlorogenic acid isomer of this comparative example was prepared using the following method: (1) Take 10 mg each of Pt / C (5 wt%), Pd / C (5 wt%) and Ru / C (5 wt%), add 4.0 mg of chlorogenic acid and 1.0 mL of D2O, and place them in a PTFE-lined pressure vessel.

[0071] (2) React at 100℃ for 24 hours.

[0072] (3) After the reaction is complete, filter to remove the catalyst, concentrate under reduced pressure, and analyze the deuteration rate.

[0073] Experimental results: No significant deuteration was detected in any of the noble metal catalysts, and DI < 5%.

[0074] Comparative Example 6: The preparation method of the deuterated chlorogenic acid isomer in this comparative example is basically the same as that in comparative example 4, except that D2O in step (1) is replaced with CD3OD.

[0075] Experimental results: DI was <5% in all cases, indicating no significant deuteration.

[0076] Comparative Example 7: The deuterated chlorogenic acid isomer of this comparative example was prepared using the following method: (1) Dissolve 4.0 mg of chlorogenic acid in 1.0 mL of D2O and add DCl to 10 mmol / L.

[0077] (2) The reaction is carried out by heating at 100°C (continuous heating).

[0078] Experimental results: Chlorogenic acid was completely degraded within minutes, and no deuterated products were detected (DI≈0%).

[0079] Example 11: This embodiment uses the deuterated chlorogenic acid isotope prepared in Example 1 as a stable isotope internal standard for LC-MS / MS quantitative analysis of chlorogenic acid in complex matrices, including the following steps: (1) Preparation of internal standard solution and standard curve Accurately weigh the deuterated chlorogenic acid isomer from Example 1, dissolve and dilute it with methanol to prepare an internal standard working solution with a concentration of 12 μg / mL. Separately, prepare a series of standard working solutions with methanol using chlorogenic acid reference standard. Add different concentrations of chlorogenic acid standard solution and a constant concentration of internal standard solution to blank human plasma. After protein precipitation with acetonitrile, inject the supernatant to obtain plasma matrix spiking curve samples with concentrations of 0.0625, 0.125, 0.25, 0.5, and 1.0 μg / mL.

[0080] (2) LC-MS / MS analysis conditions A Wondasil C18 column (4.6 × 250 mm, 5 μm) was used with gradient elution of 0.1% formic acid aqueous solution (phase A) and methanol (phase B) as the mobile phase (0–8 min, 14%–19% B; 8–14 min, 19% B; 14–34 min, 19%–31% B; 34–35 min, 31%–90% B; 35–40 min, 90% B), at a flow rate of 0.7 mL / min and a column temperature of 30 °C. Mass spectrometry was performed in negative ion electrospray spectroscopy mode with multiple reaction monitoring (MRM) channels: chlorogenic acid m / z 353→191 (quantitative) and 179 (qualitative); deuterated internal standard d3-CGA m / z 356→182, d4-CGA m / z 357→183.

[0081] (3) Matrix effect correction effect The same concentration of chlorogenic acid was added to blank plasma, and the determination was performed using both the external standard method without an internal standard and the internal standard method with an internal standard. The results showed that the recovery rate of the external standard method was only 62.46% due to the matrix inhibition effect, while the recovery rate of the internal standard method increased to 98.3% after correction, indicating that the deuterated internal standard can effectively counteract the ion inhibition effect caused by the plasma matrix.

[0082] (4) Methodological validation results Within the concentration range of 0.0625–1 μg / mL, the peak area ratio of chlorogenic acid to the internal standard showed a good linear relationship with the concentration, with a correlation coefficient R² = 0.9993. The relative standard deviations (RSDs) of intra-day and inter-day precision were both <15%, and the spiked recoveries were in the range of 85%–115%, meeting the methodological requirements for trace bioanalysis.

[0083] (5) Actual sample determination This method was applied to the determination of chlorogenic acid content in extracts of honeysuckle and wild honeysuckle. The internal standard method yielded 0.36±0.02 mg / g in honeysuckle and 0.44±0.03 mg / g in wild honeysuckle; the external standard method yielded 0.42±0.05 mg / g and 0.51±0.06 mg / g, respectively. The relative deviation of the internal standard method decreased from 13.6%~15.9% for the external standard method to 4.2%~5.7%, indicating a significant improvement in stability. In samples containing isomers such as neochlorogenic acid, the internal standard method can effectively distinguish the target peak from interfering peaks, resolving the quantitative deviation problem caused by co-effluents.

[0084] The above embodiments are only used to illustrate the technical solutions disclosed in this invention, and are not intended to limit it. Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions disclosed in the embodiments of this invention, and should all be included within the protection scope disclosed in this invention.

Claims

1. A deuterated chlorogenic acid isomer, characterized in that, The deuterated chlorogenic acid isotopes are mixtures of chlorogenic acid isotopes containing different numbers of deuterium atoms. The main components of the mixture include CA-d3 and CA-d4, and the mass-to-charge ratios of both CA-d3 and CA-d4 exceed those of the natural chlorogenic acid. 13 The C isotope envelope range is defined, and there is no isotope overlap or interference between the CA-d3 and CA-d4 multiple reaction monitoring channels and the analysis channel for undeuterated chlorogenic acid.

2. The deuterated chlorogenic acid isoform according to claim 1, characterized in that, Based on the normalized peak area calculation of negative ion electrospray ionization mass spectrometry, the combined peak area of ​​CA-d3 and CA-d4 accounts for ≥50% of the total chlorogenic acid-related peak area.

3. The deuterated chlorogenic acid isoform according to claim 1 or 2, characterized in that, In the structure of the deuterated chlorogenic acid isomer, the deuterium atom is located only in the caffeoyl part of the chlorogenic acid molecule, and the quinic acid ring does not contain a deuterium atom.

4. A method for preparing the deuterated chlorogenic acid isoform according to any one of claims 1-3, characterized in that, The process includes the following steps: dissolving chlorogenic acid in a deuterium-containing substrate, heating it in a closed container in the presence of anhydrous aluminum trichloride to carry out a hydrogen-deuterium exchange reaction, removing the solvent after the reaction is complete, and obtaining the deuterated chlorogenic acid isoform.

5. The method for preparing the deuterated chlorogenic acid isoform according to claim 4, characterized in that, The heating is done intermittently, that is, the reaction system is heated to 80-120°C and held for 10-20 minutes, then the heating is stopped and the system is allowed to cool naturally for 30-60 minutes. The above operation is repeated 2-5 times.

6. The method for preparing the deuterated chlorogenic acid isoform according to claim 5, characterized in that, The reaction system was heated to 100°C and held for 13 minutes. Then the heating was stopped and the system was allowed to cool naturally for 50 minutes. The above operation was repeated 3 times.

7. The method for preparing the deuterated chlorogenic acid isoform according to any one of claims 4-6, characterized in that, The concentration of anhydrous aluminum trichloride in the reaction system is 5–20 mmol / L.

8. The method for preparing the deuterated chlorogenic acid isoform according to any one of claims 4-6, characterized in that, The concentration of chlorogenic acid in deuterated methanol is 1–20 mg / mL.

9. The method for preparing the deuterated chlorogenic acid isoform according to claim 8, characterized in that, The concentration of chlorogenic acid in the deuterium-containing substrate is 4 mg / mL.

10. The method for preparing the deuterated chlorogenic acid isoform according to any one of claims 4-6, characterized in that, The deuterium-containing substrate includes at least one of heavy water, deuterated acetonitrile, deuterated hydrochloric acid, deuterated methanol, and deuterated acetone; the deuterated methanol is anhydrous CD3OD with a deuterium atom abundance ≥99.8 atom.

11. The method for preparing the deuterated chlorogenic acid isoform according to any one of claims 4-6, characterized in that, After the reaction is complete, the solvent is removed by concentration under reduced pressure, without column chromatography or preparative high-performance liquid chromatography purification.

12. The method for preparing the deuterated chlorogenic acid isoform according to any one of claims 4-6, characterized in that, In the prepared deuterated chlorogenic acid isomer, the combined peak area of ​​CA-d3 and CA-d4 accounts for ≥92.7% of the total chlorogenic acid-related peak area, and the substrate recovery rate is ≥88.3%.

13. The method for preparing the deuterated chlorogenic acid isoform according to any one of claims 4-6, characterized in that, In the deuterated chlorogenic acid isomers prepared in different batches, the relative standard deviation of the peak area ratio of CA-d3 to CA-d4 between batches is ≤5%.

14. The application of the deuterated chlorogenic acid isoform according to any one of claims 1-3, characterized in that, The deuterated chlorogenic acid isomer is used as a stable isotope internal standard in quantitative analysis by liquid chromatography-tandem mass spectrometry.

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