A qualitative analysis method for isomers of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid

Abstract

The invention discloses a method for qualitative analysis of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers, and belongs to the field of separation and analytical chemistry. Monosubstituted and disubstituted hydroxybenzoylquinic acid different position isomers and hydroxycinnamoylquinic acid different position isomers are synthesized through synthesis reaction, and then the secondary mass spectrometry fragmentation law and retention time law are studied, and a method for distinguishing position isomers is provided, thereby providing a new idea for qualitative analysis of isomers without standard samples.

Description

Technical Field

[0001] The invention belongs to the field of separation and analytical chemistry, and in particular relates to a qualitative analysis method for hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In the IUPAC system, quinic acid is defined as 1L-1(OH),3,4 / 5-tetrahydroxycyclohexanecarboxylic acid, which is a compound with multiple hydroxy groups and multiple esterification sites in quinic acid. Among them, hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid are phenolic acid compounds formed by esterification of quinic acid with different numbers of benzoic acid or cinnamic acid. This type of substance is widely found in plants and has antioxidant, anti-cancer, anti-inflammatory, antibacterial, and anti-diabetic effects. Hydroxycinnamoylquinic acid and hydroxybenzoylquinic acid have complex structures due to the different esterification positions and the number of acyl groups. Due to the different esterification positions, hydroxycinnamoyl and hydroxybenzoylquinic acid compounds exist as 1-, 3-, 4-, and 5-acyl isomers. According to the number of acylation groups, they can be divided into four categories, including monosubstituted, disubstituted, trisubstituted, and tetrasubstituted esters. Such compounds have complex structures, and most of them lack standard samples, which in turn brings great difficulties to compound analysis, especially the accurate identification of isomers. Therefore, there is an urgent need for a method that can accurately identify isomers of such compounds without standard samples. Summary of the invention

[0004] In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a method for qualitative analysis of isomers of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid. Hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid are synthesized by the present invention, and their MS / MS fragmentation rules and chromatographic retention rules are analyzed by a UPLC-QTOF-MS / MS system to distinguish the isomers of hydroxybenzoyl and hydroxycinnamoylquinic acid.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] On the one hand, a method for qualitative analysis of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers is characterized in that it comprises the following steps:

[0007] (1) A series of mono-substituted and di-substituted hydroxybenzoylquinic acid isomers and hydroxycinnamoylquinic acid isomers were synthesized by Steglich esterification reaction;

[0008] (2) qualitative analysis of the different positional isomers obtained in (1) by UPLC–QTOF–MS / MS;

[0009] Based on the elution patterns of caffeoylquinic acid isomers, feruloylquinic acid isomers and dicaffeoylquinic acid isomers with standard samples, the chromatographic retention patterns of the other mono-substituted and di-substituted hydroxybenzoylquinic acid isomers at different positions and hydroxycinnamoylquinic acid isomers at different positions were determined.

[0010] The eluted isomers at different positions were subjected to secondary mass spectrometry analysis in negative ion mode with multiple collision energies, and qualitative judgments were made on the isomers at different positions based on the types or relative intensities of the main fragment ions of the isomers or the intensity ratios of the daughter ions and the parent ions.

[0011] The beneficial effects of the present invention are:

[0012] Except for caffeoylquinic acid and dicaffeoylquinic acid, and some isomers of monoferuloylquinic acid, there are no standards for diferuloylquinic acid, p-coumaroylquinic acid, di-p-coumaroylquinic acid, vanillylquinic acid, divanillylquinic acid, syringoylquinic acid and divanillylquinic acid isomers. Therefore, there is no way to study the mass spectrometry rules of them, and no one has studied it before. The invention synthesizes these mono-substituted and di-substituted hydroxybenzoylquinic acid different position isomers and hydroxycinnamoylquinic acid different position isomers through a synthesis reaction, uses the elution rules of caffeoylquinic acid and dicaffeoylquinic acid with standard samples as a basis, confirms the chromatographic retention rules of the other mono-substituted and di-substituted hydroxybenzoylquinic acid different position isomers and hydroxycinnamoylquinic acid different position isomers, and uses part of monoferuloylquinic acid with standard samples for verification to confirm accuracy; then, the secondary mass spectrometry fragmentation rules and are studied, and a method for distinguishing the position isomers is provided, thereby providing a new idea for the qualitative analysis of isomers without standard samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 It is the secondary mass spectrum of monovanillylquinic acid isomer;

[0015] Figure 2 is the secondary mass spectrum of monosyringoylquinic acid isomer;

[0016] Figure 3 is the secondary mass spectrum of the monocaffeoylquinic acid isomer;

[0017] Figure 4 is the secondary mass spectrum of the monoferuloylquinic acid isomer;

[0018] Figure 5 It is the secondary mass spectrum of single p-coumaroylquinic acid isomer;

[0019] Figure 6 is the secondary mass spectrum of dicaffeoylquinic acid isomers;

[0020] Figure 7 is the secondary mass spectrum of diferuloylquinic acid isomers;

[0021] Figure 8 It is the secondary mass spectrum of di-p-coumarylquinic acid isomers;

[0022] Fig. 9 This is the secondary mass spectrum of divanillylquinic acid isomers;

[0023] Fig.10 This is the secondary mass spectrum of disyringylquinic acid isomers. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0026] Since there are no standard samples for isomers of diferuloylquinic acid, p-coumaroylquinic acid, vanillylquinic acid and syringoylquinic acid, and there is no way to study their mass spectrometry rules, the present invention proposes a qualitative analysis method for isomers of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid.

[0027] A typical embodiment of the present invention provides a method for qualitative analysis of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers, comprising the following steps:

[0028] (1) A series of mono-substituted and di-substituted hydroxybenzoylquinic acid isomers and hydroxycinnamoylquinic acid isomers were synthesized by Steglich esterification reaction;

[0029] (2) qualitative analysis of the different positional isomers obtained in (1) by UPLC–QTOF–MS / MS;

[0030] Based on the elution patterns of caffeoylquinic acid isomers, feruloylquinic acid isomers and dicaffeoylquinic acid isomers with standard samples, the chromatographic retention patterns of the other mono-substituted and di-substituted hydroxybenzoylquinic acid isomers at different positions and hydroxycinnamoylquinic acid isomers at different positions were determined.

[0031] The eluted isomers at different positions were subjected to secondary mass spectrometry analysis in negative ion mode with multiple collision energies, and qualitative judgments were made on the isomers at different positions based on the types or relative intensities of the main fragment ions of the isomers or the intensity ratios of the daughter ions and the parent ions.

[0032] In some examples of this implementation mode, the confirmation of the chromatographic retention rule is specifically: the chromatographic retention rules of the 3-, 4-, 5-position monocaffeoylquinic acid isomers and the monoferuloylquinic acid isomers of the standard samples are consistent, thereby determining that the chromatographic retention rules of the other mono-substituted and di-substituted hydroxybenzoylquinic acid isomers at different positions and the hydroxycinnamoylquinic acid isomers at different positions are also consistent.

[0033] In some examples of this embodiment, the hydroxybenzoylquinic acid includes vanillylquinic acid, syringylquinic acid, divanillylquinic acid, and disyringylquinic acid.

[0034] In some examples of this embodiment, the hydroxycinnamoylquinic acid includes feruloylquinic acid, p-coumaroylquinic acid, diferuloylquinic acid, and di-p-coumaroylquinic acid.

[0035] In some examples of this embodiment, the collision energy is 10-30 eV.

[0036] In some examples of this embodiment, the chromatographic conditions are: the chromatographic column is a 2.1 mm×100 mm, 1.8 μm Agilent SB-C18 column, the flow rate is 0.3 mL / min, the column temperature is 40° C., and the injection volume is 2 μL.

[0037] In some examples of this embodiment, mobile phases A and B are respectively an aqueous solution and an acetonitrile solution containing 0.6% by mass of formic acid;

[0038] The elution procedure was gradient elution: the initial gradient was 5% B; after 20 min, it was linearly increased to 25% B; after 35 min, it was linearly increased to 100% B and maintained for 5 min; finally, it was reduced to 5% B and maintained for 5 min.

[0039] In some examples of this embodiment, the mass spectrometry conditions are: nebulizer, 2.0 Bar; drying gas flow rate, 8 L / min; nebulizing gas pressure, 200 kPa; drying gas temperature, 200°C; ion funnel, 300 Vpp; hexapole, 60 Vpp; transmission time, 70 μs; collision cell RF voltage amplitude, 600 Vpp; pre-pulse waiting time, 7 μs; capillary voltage is set to 3000 V in negative mode; mass number scanning range is 50-1200; collision energy is 10-30 eV during secondary mass spectrometry analysis.

[0040] In some examples of this implementation mode, the Steglich esterification reaction is as follows: in a reaction container, benzoic acid or cinnamic acid, quinic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-lutidine are added in sequence, under the protection of an inert gas, an organic solvent is added, the reaction is stirred at room temperature, and after the reaction is completed, the reaction is filtered and vacuum dried to obtain the product.

[0041] In some examples of this embodiment, the molar ratio of hydroxybenzoic acid or hydroxycinnamic acid to quinic acid, 1-ethyl-(3-dimethylanilinepropyl)carbodiimide hydrochloride and 4-lutidine is 1-1.5:1-1.5:1-1.5:0.5-1, preferably 1:1.5:1.5:0.5.

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0043] Example 1

[0044] A qualitative analysis method for hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers, comprising:

[0045] 1) Synthesis of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers: A series of mono- or di-substituted hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid are synthesized by Steglich esterification of caffeic acid, ferulic acid, p-coumaric acid, vanillic acid or syringic acid (a) with quinic acid (b) catalyzed by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-lutidine (DMAP).

[0046] Specifically: in a 50mL round-bottom flask, add a (1mmol, 1eq), b (1.5mmol, 1.5eq), EDCI (1.5mmol, 1.5eq) and DMAP (0.5mmol, 0.5eq) in sequence, under nitrogen protection, add 10mL of dichloromethane solvent, stir and react at room temperature for 6h, filter, wash the filter cake with petroleum ether and ethyl acetate three times in sequence, and then dry in vacuo at 60°C for 12h to obtain the reactant.

[0047] 2) Liquid chromatography-mass spectrometry analysis conditions: Ultra-high performance liquid chromatography (UPLC, ACQUITY, H-Class, Waters)-high-resolution time-of-flight mass spectrometry (Impact II, Bruker) was used.

[0048] The chromatographic column was an Agilent SB-C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent, USA), the flow rate was 0.3 mL / min, the column temperature was 40°C, and the injection volume was 2 μL.

[0049] The mobile phases A and B were aqueous solution and acetonitrile solution containing 0.6% by mass of formic acid, respectively.

[0050] Elution gradient: Initial gradient was 5% B; after 20 minutes, it was linearly increased to 25% B; after 35 minutes, it was linearly increased to 100% B and maintained for 5 minutes; finally, it was reduced to 5% B and maintained for 5 minutes. The total time was 45 minutes.

[0051] The mass spectrometry conditions were as follows: nebulizer, 2.0 Bar; drying gas flow rate, 8 L / min; nebulizing gas pressure, 200 kPa; drying gas temperature, 200 °C; ion funnel, 300 Vpp; hexapole, 60 Vpp; transmission time, 70 μs; collision cell RF voltage amplitude, 600 Vpp; pre-pulse waiting time, 7 μs; capillary voltage set to 3000 V in negative mode; mass number scanning range of 50-1200; collision energy of 10-30 eV for secondary mass spectrometry analysis. The synthesized reactants were subjected to MS / MS analysis, and the secondary collision energy was set to 10-30 eV.

[0052] 3) Elution patterns of hydroxycinnamoylquinic acid and hydroxybenzoylquinic acid isomers: The elution patterns of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers were studied.

[0053] Based on the elution rules of caffeoylquinic acid isomers, feruloylquinic acid, and dicaffeoylquinic acid isomers with standard samples, the chromatographic retention rules of the remaining mono-substituted and di-substituted hydroxybenzoylquinic acid isomers at different positions and hydroxycinnamoylquinic acid isomers at different positions were determined. The rules are as follows:

[0054] 1. Monosubstituted hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid

[0055] The elution order of monohydroxycinnamoylquinic acid and hydroxybenzoylquinic acid on the C18 reverse phase column was the same.

[0056] The elution order of caffeoylquinic acid isomers on the C18 reverse phase chromatography column was 1-O-caffeoylquinic acid (3.1 min) > 3-O-caffeoylquinic acid (4.3 min) > 5-O-caffeoylquinic acid (7.8 min) > 4-O-caffeoylquinic acid (8.4 min).

[0057] The elution order of feruloylquinic acid isomers on the C18 reverse phase chromatography column was 1-O-feruloylquinic acid (6.9 min) > 3-O-feruloylquinic acid (7.9 min) > 5-O-feruloylquinic acid (11.2 min) > 4-O-feruloylquinic acid (11.3 min).

[0058] The elution order of vanillylquinic acid isomers on the C18 reverse phase chromatography column was 1-O-vanillylquinic acid (2.5 min) > 3-O-vanillylquinic acid (3.0 min) > 5-O-vanillylquinic acid (6.8 min) > 4-O-vanillylquinic acid (6.9 min).

[0059] The elution order of syringoylquinic acid isomers on the C18 reverse phase chromatography column was 1-O-syringoylquinic acid (3.4 min) > 3-O-syringoylquinic acid (4.0 min) > 5-O-syringoylquinic acid (7.8 min) > 4-O-syringoylquinic acid (8.3 min).

[0060] The elution order of p-coumaroylquinic acid isomers on the C18 reverse phase chromatography column was 1-O-p-coumaroylquinic acid (4.7 min) > 3-O-p-coumaroylquinic acid (6.1 min) > 5-O-p-coumaroylquinic acid (9.8 min) > 4-O-p-coumaroylquinic acid (10.0 min).

[0061] 2. Disubstituted hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid

[0062] The elution order of dihydroxycinnamoylquinic acid and dihydroxybenzoylquinic acid on the C18 reverse phase column was the same.

[0063] The elution order of dicaffeoylquinic acid on the C18 reverse phase chromatographic column was 1,3-O-caffeoylquinic acid (11.2 min) > 1,4-O-caffeoylquinic acid (16.4 min) > 3,4-O-caffeoylquinic acid (16.7 min) > 1,5-O-caffeoylquinic acid (17.2 min) > 3,5-O-caffeoylquinic acid (17.3 min) > 4,5-O-caffeoylquinic acid (18.7 min).

[0064] The elution order of diferuloylquinic acid on the C18 reverse phase chromatography column was 1,3-O-feruloylquinic acid (16.5 min) > 1,4-O-feruloylquinic acid (22.7 min) > 3,4-O-feruloylquinic acid (23.2 min) > 1,5-O-feruloylquinic acid (23.7 min) > 3,5-O-feruloylquinic acid (23.8 min) > 4,5-O-feruloylquinic acid (24.6 min).

[0065] The elution order of di-p-coumaroylquinic acid on the C18 reverse phase chromatographic column was 1,3-O-p-coumaroylquinic acid (16.8 min) > 1,4-O-p-coumaroylquinic acid (21.1 min) > 3,4-O-p-coumaroylquinic acid (22.0 min) > 1,5-O-p-coumaroylquinic acid (23.2 min) > 3,5-O-p-coumaroylquinic acid (23.4 min) > 4,5-O-p-coumaroylquinic acid (24.2 min).

[0066] The elution order of divanillylquinic acid on the C18 reverse phase chromatography column was 1,3-O-vanillylquinic acid (9.4 min) > 1,4-O-vanillylquinic acid (11.0 min) > 3,4-O-vanillylquinic acid (16.5 min) > 1,5-O-vanillylquinic acid (17.6 min) > 3,5-O-vanillylquinic acid (18.5 min) > 4,5-O-vanillylquinic acid (21.7 min).

[0067] The elution order of disyringoylquinic acid on the C18 reverse phase chromatographic column was 1,3-O-syringoylquinic acid (8.6 min) > 1,4-O-syringoylquinic acid (13.4 min) > 3,4-O-syringoylquinic acid (17.0 min) > 1,5-O-syringoylquinic acid (18.0 min) > 3,5-O-syringoylquinic acid (18.9 min) > 4,5-O-syringoylquinic acid (21.5 min).

[0068] 4) Mass spectrometric fragmentation rules of hydroxycinnamoylquinic acid and hydroxybenzoylquinic acid isomers: The MS / MS spectra of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid at different collision energies in negative ion mode were analyzed, and the correlation analysis of the relative intensities of the main fragment ions of the isomers was performed. The isomers at different positions were qualitatively judged according to the types or relative intensities of the main fragment ions of the isomers or the intensity ratios of the daughter ions and the parent ions.

[0069] 1. Monosubstituted hydroxybenzoylquinic acid

[0070] Vanillylquinic acid is formed by the esterification of one molecule of vanillic acid and quinic acid. The MS / MS spectra of 1-O-vanillylquinic acid, 3-O-vanillylquinic acid, 4-O-vanillylquinic acid and 5-O-vanillylquinic acid are significantly different. Figure 1 ), the base peak of 3-O-vanillylquinic acid is 167. The base peak of 5-O-vanillylquinic acid is 191. The base peak of 4-O-vanillylquinic acid is 173. In 1-O-vanillylquinic acid, the intensities of 167 and 191 are both relatively high. The peak intensity of 191 is 92% of the base peak 167.

[0071] Syringoylquinic acid is formed by the esterification of one molecule of syringic acid and quinic acid. The MS / MS spectra of 1-O-syringoylquinic acid, 3-O-syringoylquinic acid, 4-O-syringoylquinic acid and 5-O-syringoylquinic acid are significantly different. Figure 2 ), the base peak of 3-O-syringoylquinic acid is 197. The base peak of 5-O-syringoylquinic acid is 191. The base peak of 4-O-syringoylquinic acid is 173. In 1-O-vanillylquinic acid, the intensities of 197 and 173 are relatively high, and the peak intensity of 173 is 74% of the base peak 197.

[0072] 2. Monosubstituted hydroxycinnamoylquinic acid

[0073] Caffeoylquinic acid is formed by the esterification of one molecule of caffeic acid and quinic acid. The MS / MS spectra of 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid are significantly different. Figure 3 ), the base peak of 3-O-caffeoylquinic acid is 191, and the peak intensity of 179 is 77% of the base peak. The base peak of 5-O-caffeoylquinic acid is 191. The base peak of 4-O-caffeoylquinic acid is 173, and the relative peak intensity of 179 is 79% of the base peak. The base peak of 1-O-caffeoylquinic acid is 191.

[0074] Feruloylquinic acid is formed by the esterification of one molecule of ferulic acid and quinic acid. The MS / MS spectra of 1-O-feruloylquinic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid and 5-O-feruloylquinic acid are significantly different. Figure 4 ), the base peak of 3-O-feruloylquinic acid is 193. The base peak of 5-O-feruloylquinic acid is 191. The base peak of 4-O-feruloylquinic acid is 173. The base peak of 1-O-caffeoylquinic acid is 193, and the peak intensity of 173 is 68% of the base peak.

[0075] p-Coumarylquinic acid is formed by the esterification of p-coumaric acid and quinic acid. The MS / MS spectra of 1-O-p-coumarylquinic acid, 3-O-p-coumarylquinic acid, 4-O-p-coumarylquinic acid and 5-O-p-coumarylquinic acid are significantly different. Figure 5 ), the base peak of 3-O-p-coumarylquinic acid is 163. The base peak of 5-O-p-coumarylquinic acid is 191, and the peak intensity of 173 is 60% of the base peak. The base peak of 4-O-p-coumarylquinic acid is 173. The base peak of 1-O-p-coumarylquinic acid is 191.

[0076] 3. Disubstituted hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid

[0077] Dicaffeoylquinic acid is formed by the esterification of two molecules of caffeic acid and quinic acid. Under high collision energy, the base peaks of 1,4-O-caffeoylquinic acid, 4,5-O-caffeoylquinic acid, and 3,4-O-caffeoylquinic acid are 173, while the base peaks of 1,3-O-caffeoylquinic acid, 1,5-O-caffeoylquinic acid, and 3,5-O-caffeoylquinic acid are 191. As the collision energy increases, the signal intensity of the parent ion 515 gradually weakens, and the signal intensity of the daughter ion 353 gradually increases. The intensity ratio of the daughter ion 353 to the parent ion 515 (353 / 515) can further distinguish the above isomers. At 15 and 20 eV collision energies ( Figure 6 ), the intensity ratios of daughter ion 353 and parent ion 515 (353 / 515) in 1,4-O-caffeoylquinic acid, 4,5-O-caffeoylquinic acid, and 3,4-O-caffeoylquinic acid are in the order of 1,4>4,5>3,4. The intensity ratios of daughter ion 353 and parent ion 515 (353 / 515) in 1,3-O-caffeoylquinic acid, 1,5-O-caffeoylquinic acid, and 3,5-O-caffeoylquinic acid are in the order of 3,5-O-caffeoylquinic acid>1,5-O-caffeoylquinic acid>1,3-O-caffeoylquinic acid.

[0078] Diferuloylquinic acid is formed by the esterification of two molecules of ferulic acid and quinic acid. At a collision energy of 15 eV, the base peaks of 1,4-O-feruloylquinic acid and 3,4-O-feruloylquinic acid are 349, while the base peaks of 1,3-O-feruloylquinic acid, 1,5-O-feruloylquinic acid, 3,5-O-feruloylquinic acid, and 4,5-O-feruloylquinic acid are 367. With the increase of collision energy, the signal intensity of parent ion 543 gradually weakens, and the signal intensity of daughter ion 367 gradually increases. The intensity ratio (367 / 543) of daughter ion 367 and parent ion 543 can further distinguish the above isomers. At a collision energy of 15 and 20 eV ( Figure 7 ), the intensity ratio of daughter ion 367 to parent ion 543 (367 / 543) is higher in 3,4-O-feruloylquinic acid than in 1,4-O-feruloylquinic acid. The intensity ratio of daughter ion 353 to parent ion 515 (353 / 515) in 1,3-O-feruloylquinic acid, 1,5-O-feruloylquinic acid, 3,5-O-feruloylquinic acid, 4,5-O-feruloylquinic acid is in the order of 1,5-O-feruloylquinic acid>1,3-O-feruloylquinic acid>4,5-O-feruloylquinic acid>3,5-O-feruloylquinic acid.

[0079] Di-p-coumarylquinic acid is formed by the esterification of two molecules of p-coumaric acid and quinic acid. Under high collision energy, the base peak of 1,4-O-p-coumarylquinic acid is 173; the base peaks of 1,3-O-p-coumarylquinic acid and 3,5-O-p-coumarylquinic acid are 163; and the base peaks of 3,4-O-p-coumarylquinic acid, 1,5-O-p-coumarylquinic acid, and 4,5-O-p-coumarylquinic acid are 191. With the increase of collision energy, the signal intensity of parent ion 483 gradually weakens, and the signal intensity of daughter ion 337 gradually increases. The intensity ratio of daughter ion 337 to parent ion 483 (337 / 483) can further distinguish the above isomers. At 15 and 20 eV collision energies ( Figure 8 ), the intensity ratio of daughter ion 337 to parent ion 483 (337 / 483) is higher in 1,3-O-p-coumarylquinic acid than in 3,5-O-p-coumarylquinic acid. The intensity ratio of daughter ion 337 to parent ion 483 (337 / 483) in 3,4-O-p-coumarylquinic acid, 1,5-O-p-coumarylquinic acid, and 4,5-O-p-coumarylquinic acid is 3,4-O-p-coumarylquinic acid>1,5-O-p-coumarylquinic acid>4,5-O-p-coumarylquinic acid.

[0080] Divanillylquinic acid is formed by the esterification of two molecules of vanillic acid and quinic acid. Fig. 9), the base peaks of 1,3-O-vanillylquinic acid, 3,4-O-vanillylquinic acid, and 1,5-O-vanillylquinic acid are 323, and the intensity ratios of the daughter ion 323 and the parent ion 491 (323 / 491) are in the order of 1,5-O-vanillylquinic acid>1,3-O-vanillylquinic acid>3,4-O-vanillylquinic acid. The base peaks of 1,4-O-vanillylquinic acid and 3,5-O-vanillylquinic acid are 317, and the intensity ratios of the daughter ion 317 and the parent ion 491 (317 / 491) are higher in 3,5-O-vanillylquinic acid than in 1,4-O-vanillylquinic acid. However, the base peak of 4,5-O-vanillylquinic acid is 491. In addition, compared with other divanillylquinic acids, 1,3-O-vanillylquinic acid and 3,4-O-vanillylquinic acid do not have a signal ion of 191.

[0081] Disyringylquinic acid is formed by the esterification of two molecules of syringic acid and quinic acid. Fig.10 ), the base peak of 1,3-O-syringoylquinic acid, 3,4-O-syringoylquinic acid, and 1,5-O-syringoylquinic acid is 353, and the order of the intensity ratio of the daughter ion 353 to the parent ion 551 (353 / 551) is 1,5-O-syringoylquinic acid>3,4-O-syringoylquinic acid>1,3-O-syringoylquinic acid. The base peak of 1,4-O-vanillylquinic acid, 3,5-O-vanillylquinic acid, and 4,5-O-syringoylquinic acid is 377, and the order of the intensity ratio of the daughter ion 377 to the parent ion 551 (377 / 551) is 3,5-O-vanillylquinic acid>1,4-O-vanillylquinic acid>4,5-O-syringoylquinic acid. In addition, compared with other disyringylquinic acids, 1,3-O-syringylquinic acid, 3,4-O-syringylquinic acid and 4,5-O-syringylquinic acid do not have a signal ion of 191.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A qualitative analysis method for hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers, characterized in that: The following steps are involved: (1) A series of mono-substituted and di-substituted hydroxybenzoylquinic acid isomers and hydroxycinnamoylquinic acid isomers were synthesized by Steglich esterification reaction; (2) Qualitative analysis of the different positional isomers obtained in (1) by UPLC-QTOF-MS / MS; Based on the elution patterns of caffeoylquinic acid isomers, feruloylquinic acid isomers and dicaffeoylquinic acid isomers with standard samples, the chromatographic retention patterns of the other mono-substituted and di-substituted hydroxybenzoylquinic acid isomers at different positions and hydroxycinnamoylquinic acid isomers at different positions were determined. The different positional isomers obtained by elution were subjected to secondary mass spectrometry analysis with multiple collision energies in negative ion mode. The different positional isomers of monosubstituted hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid were qualitatively judged according to the main fragment ion types and relative intensities of the isomers. The different positional isomers of disubstituted hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid were qualitatively judged according to the intensity ratio of daughter ions and parent ions. The confirmation of the chromatographic retention law is specifically as follows: the chromatographic retention laws of the 3-, 4-, 5-position monocaffeoylquinic acid isomers and monoferuloylquinic acid isomers of the standard samples are consistent, thereby confirming that the chromatographic retention laws of the other mono-substituted and di-substituted hydroxybenzoylquinic acid isomers at different positions and hydroxycinnamoylquinic acid isomers at different positions are also consistent; The hydroxybenzoylquinic acid includes vanillylquinic acid, syringylquinic acid, divanillylquinic acid, disyringylquinic acid; The hydroxycinnamoylquinic acid includes feruloylquinic acid, p-coumarylquinic acid, diferuloylquinic acid, di-p-coumarylquinic acid; The collision energy is 10-30 eV; The chromatographic conditions were as follows: 2.1 mm × 100 mm, 1.8 μm Agilent SB-C18 column, flow rate 0.3 mL / min, column temperature 40 °C, injection volume 2 μL; Mobile phases A and B were aqueous solution and acetonitrile solution containing 0.6% formic acid by mass, respectively; The elution procedure was gradient elution: the initial gradient was 5% B; after 20 min, it was linearly increased to 25% B; after 35 min, it was linearly increased to 100% B and maintained for 5 min; finally, it was reduced to 5% B and maintained for 5 min; the total time was 45 min; The mass spectrometry conditions were: nebulizer, 2.0 Bar; dry gas flow rate, 8 L / min; Atomizing gas pressure, 200 kPa; drying gas temperature, 200 ˚C; ion funnel, 300 Vpp; Hexapole, 60 Vpp; transit time, 70 μs; The RF voltage amplitude of the collision cell was 600 Vpp; the waiting time before pulse was 7 μs; the capillary voltage was set to 3000 V in negative mode; the mass number scanning range was 50-1200; and the collision energy was 10-30 eV during secondary mass spectrometry analysis.

2. The method for qualitative analysis of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers according to claim 1, characterized in that: The Steglich esterification reaction is as follows: benzoic acid or cinnamic acid, quinic acid, 1-ethyl-(3-dimethylanilinepropyl)carbodiimide hydrochloride and 4-lutidine are added in sequence in a reaction container, an organic solvent is added under the protection of an inert gas, the reaction is stirred at room temperature, and after the reaction is completed, the product is filtered and vacuum dried to obtain the product.

3. The method for qualitative analysis of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers according to claim 1, characterized in that: The molar ratio of hydroxybenzoic acid or hydroxycinnamic acid to quinic acid, 1-ethyl-(3-dimethylanilinepropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 1-1.5:1-1.5:1-1.5:0.5-1.

4. The method for qualitative analysis of hydroxybenzoylquinic acid and hydroxycinnamoylquinic acid isomers as claimed in claim 3, characterized in that: The molar ratio of hydroxybenzoic acid or hydroxycinnamic acid to quinic acid, 1-ethyl-(3-dimethylanilinepropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1.5:1.5:0.5.

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