Targeted identification method of crocin compounds and new crocin compounds
By using a combination method of multi-stage high-resolution mass spectrometry and targeted parent ion list in the identification of saffron compounds, the problem of identification of saffron compounds in the prior art was solved, and the rapid isolation and identification of saffron compounds and the discovery of new compounds were achieved.
Patent Information
- Application Number
- CN202110565200.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-05-24
- Publication Date
- 2025-04-29
- Estimated Expiration
- 2041-05-24
AI Technical Summary
The prior art is difficult to accurately isolate and identify low content and isomer compounds in saffron compounds, resulting in increased identification difficulty.
The targeted identification method based on multi-stage high-resolution mass spectrometry is adopted, and the ultraviolet absorption, precursor ion mass-charge ratio and multi-stage fragment ion mass-charge ratio are obtained by combining the targeted parent ion list with multi-stage high-resolution mass spectrometry, thereby achieving rapid separation and identification.
Rapid isolation and identification of saffron compounds were achieved, and new saffron compounds were discovered and identified, which improved the identification level of mass spectrometry detection technology.
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Figure CN115389639B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of identification of components of traditional Chinese medicine, and in particular to a targeted identification method of crocin compounds and new crocin compounds. Background Art
[0002] Crocus saffron is known as "plant gold". It is scarce and expensive, but it has high medicinal value. In addition, it is a medicinal material with medicinal and edible properties, with little toxicity and side effects. In addition to being used for the treatment of diseases, it can also play a role in daily nourishment and health care. Therefore, it has received widespread attention and is widely used in food, health products and medicine. Crocus saffron has a rich variety of chemical components, mainly including terpenes, flavonoids, phenolic acids, anthraquinones, cyclohexanes, cyclohexenes, alkaloids, etc. Among them, terpenoid compounds are the main active ingredients and have the most extensive pharmacological research literature. Among them, the diterpenoid compound "crocin" is rich in the medicinal parts of saffron, accounting for as much as 6% to 16%, and plays an active therapeutic role in research fields such as anti-inflammatory, antibacterial, antidepressant, blood pressure reduction, blood sugar reduction, and blood lipid reduction. Therefore, in-depth exploration and identification of the types and structures of crocin compounds are of great research significance for clarifying the rich pharmacological activities of saffron.
[0003] At present, the separation and identification of crocin usually adopts ultra-high performance liquid phase tandem mass spectrometry (UPLC-MS / MS). The literature [Manuel Carmona. J. Agric. Food Chem. 2006, 54, 973-979] used this method to identify 15 crocin compounds, all of which are composed of 1 to 5 glucoses connected to the cis and trans configurations of crocetin. In addition to the 15 crocin compounds most commonly reported in the literature, there may be other low-content crocin compounds, but due to the limitations of the original analytical detection technology, it is difficult to accurately separate and identify them. In addition, crocin has a variety of isomers. Since isomer molecules often produce similar fragment ions, distinguishing isomer molecules has always been a difficulty in mass spectrometry identification. Conventional secondary mass spectrometry MS 2 Due to the unclear mass spectrometry fragmentation pathway of the compound, the fragment ions cannot be accurately attributed, which brings certain difficulties to the identification of crocin compounds with the same molecular weight. Therefore, how to establish an effective separation and identification method, improve the identification level of mass spectrometry detection technology, and ultimately discover and identify more new compounds has always been the focus and difficulty of saffron mass spectrometry qualitative analysis. Summary of the invention
[0004] The problem to be solved by the present invention is to establish a targeted identification method for crocin compounds based on multi-stage high-resolution mass spectrometry, and a new crocin compound obtained based on the identification method. The method is based on the combination of a targeted parent ion list and a multi-stage high-resolution mass spectrometry to obtain the ultraviolet absorption, parent ion mass-to-charge ratio, and secondary to quaternary fragment ion mass-to-charge ratio of the crocin compound, thereby performing identification. The method is simple and highly targeted, and can achieve rapid separation and identification of crocin components in the traditional Chinese medicine saffron, and identify new crocin compounds.
[0005] The technical solution of the present invention is as follows:
[0006] On the one hand, the present invention provides a targeted identification method for crocin compounds, the specific contents of which are as follows:
[0007] The targeted identification method of crocin compounds comprises the following steps:
[0008] S1. preparing saffron extract and crocin standard;
[0009] S2. Based on the core structure of crocin and the reported structural rules of crocin compounds, the possible substituent types of crocin are inferred, and a list of targeted parent ions is generated based on the possible substituents. The list of targeted parent ions includes [MH] - 、[M+HCOO] - and [M+2HCOO] 2- Additive peak, crocin compound is generated by dehydration of crocetin with 1 to 6 glucose molecules or dehydration esterification with 1 to 2 methanol molecules;
[0010] S3, injecting the saffron extract and the crocin standard into a high performance liquid chromatography-mass spectrometer, and inputting the ions in the target parent ion list into the mass spectrometer as target substances, and obtaining the ultraviolet absorption, parent ion mass-to-charge ratio, secondary to quaternary fragment ion mass-to-charge ratio and retention time of all crocin components in the saffron extract and the crocin standard respectively;
[0011] S4. Identify known crocin compound types by comparing the UV absorbance, mass-to-charge ratio of primary to quaternary fragment ions and retention time of all crocin components in the saffron extract with the UV absorbance, parent ion mass-to-charge ratio, mass-to-charge ratio of secondary to quaternary fragment ions and retention time of crocin standards;
[0012] Combining the multi-stage cleavage pattern of crocin, cleavage fragments and corresponding UV absorption wavelengths, three new crocin compounds were identified from saffron
[0013] Preferably, in step S4, three new crocin compounds, crocin hexaglycoside, crocin tetraglycoside and monomethyl esterified crocin bioside, are identified from saffron, and the structural formulas of the three new crocin compounds are:
[0014] Crocin hexaglycoside:
[0015]
[0016] Crocin Tetrasaccharoside:
[0017]
[0018] Monomethyl esterified crocin bioside:
[0019]
[0020] Preferably, in step S2, the targeted parent ion list is specifically:
[0021]
[0022]
[0023] Preferably, in step S3, the ultra high performance liquid chromatography conditions include:
[0024] Chromatographic column: C18 column;
[0025] Mobile phase: Phase A is acetonitrile, phase B is 0.1%-0.5% formic acid water or 0.1%-0.5% acetic acid water;
[0026] Gradient elution 0-21 min, 5-95% A;
[0027] Column temperature 20-40°C;
[0028] The detection wavelength is 190-600nm.
[0029] Preferably, in step S2, the C18 chromatographic column is selected from one of CORTECS UPLC C18, ACQUITY UPLC BEH C18, ACQUITY UPLC HSS PFP, C18HCE, C18HC and Zorbax SB-C18.
[0030] Preferably, in step S3, the mass spectrometry conditions include:
[0031] ESI ion source: negative ion mode;
[0032] Evaporation temperature: 250-350℃;
[0033] Ion transfer tube temperature: 250-350℃;
[0034] Auxiliary gas: 30-40Arb;
[0035] Primary parameters: MS scanning range, m / z 100-1500;
[0036] Input the ions in the target precursor ion list into the mass spectrometer as the target substances, give priority to the precursor ions in the target precursor ion list for secondary fragmentation, then select the three most abundant ions from the obtained secondary fragments to perform tertiary fragmentation again, and finally select the most abundant ions from the obtained tertiary fragments to perform quaternary fragmentation;
[0037] Multi-level parameters: MS2~MS4 scanning range, m / z 50-1500; HCD or CID collision mode; collision energy 10-100.
[0038] Preferably, in step S1, the preparation method of saffron extract comprises: taking the ground saffron stigma powder, adding 50-100% methanol aqueous solution or 30-100% ethanol aqueous solution, ultrasonically extracting for 0.5-1.5 hours at a solid-liquid ratio of 15-25 mg:1 mL, centrifuging and taking the supernatant and diluting it to 0.5 mg / mL with 50% methanol.
[0039] Preferably, in step S1, the method for preparing the crocin standard comprises: taking crocin I, crocin II, crocin III, and crocin IV standard products respectively, and preparing them into a 0.5 μg / mL standard solution with 50% methanol.
[0040] Another aspect of the present invention provides a new crocin compound obtained by the above identification method, the specific contents of which are as follows:
[0041] A new crocin compound, the new crocin compound is crocin hexaglycoside, and its structural formula is as follows:
[0042]
[0043] A new crocin compound, the new crocin compound is crocin tetrasaccharide, and its structural formula is as follows:
[0044]
[0045] A new crocin compound, the new crocin compound is a monomethyl esterified crocin bioside, and its structural formula is as follows:
[0046]
[0047] The present invention has at least one of the following beneficial effects:
[0048] The present invention provides a separation and identification method for crocin compounds, which adopts an ultra-high performance liquid chromatography-tandem high-resolution multi-stage mass spectrometry method, sets a targeted parent ion list according to the type of substituents that crocin may be connected to, collects crocin components in a targeted manner, obtains the ultraviolet absorption spectrum, accurate molecular weight, and mass spectrometry multi-stage fragmentation (secondary to fourth level, MS 2-4 ) fragment ion information, not only can the known types of crocin compounds be identified, but also three new crocin compounds are finally discovered and identified. The identification method of the present invention realizes the rapid separation and identification of crocin components in the traditional Chinese medicine saffron, and provides a basis for further research on the pharmacological material basis of saffron. At the same time, the identification method of the present invention is simple and highly targeted, which provides an effective strategy for the discovery of new compounds.
[0049] The present invention predicts possible substituent types of crocin compounds in advance to form a targeted parent ion list, and inputs the parent ions in the list as target substances into a mass spectrometer, so that mass spectrometer acquisition data will preferentially use the parent ions in the parent ion list to generate secondary fragments. If the target substance is not input into the mass spectrometer, the mass spectrometer only selects ions for secondary generation in the order of intensity, which will cause some low-content crocin compounds to be unable to be detected. In the present invention, by inputting the parent ion list into the mass spectrometer, that is, inputting the mass-to-charge ratio of the target substance crocin, the target substance can be targeted for identification. Even if the compound intensity is very low, the mass spectrometer can preferentially select the target substance to generate secondary fragments, which is targeted and is conducive to the identification of the target substance.
[0050] The method of the present invention is used to identify three unreported new crocin compounds, crocin hexaside, crocin tetraside and monomethyl esterified crocin bioside, from saffron for the first time, thus filling the blank in the research direction of new crocin compounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0051] Figure 1 is the ultraviolet absorption graph of the chromatographic column to the saffron extract in Example 1;
[0052] Figure 2 is the mass spectrum of the crocin targeted parent ion list extraction in Example 1;
[0053] Figure 3 Schematic diagram of the multi-stage mass spectrometry fragmentation pathway of crocin I to IV standards under HCD / CID combined collision energy in Example 1;
[0054] Figure 4 This is the UV absorption diagram of the standard products of crocin I to IV in Example 1
[0055] Figure 5The multi-stage mass spectrometry fragmentation diagram and fragmentation pathway of the new crocin compound 1trans-6-(2G,4G) in Example 1;
[0056] Figure 6 The secondary mass spectrometry fragmentation diagram and fragmentation pathway of the new crocin compound 2trans-4-(1g, 3G) in Example 1;
[0057] Figure 7 The multi-stage mass spectrometry fragmentation diagram and fragmentation pathway of the new crocin compound 3trans-2G methyl ester in Example 1;
[0058] Figure 8 is the ultraviolet absorption graph of the new crocin compounds 1, 2 and 3 in Example 1;
[0059] Fig. 9 is the ultraviolet absorption graph of the chromatographic column to the saffron extract in Example 2;
[0060] Fig.10 is the ultraviolet absorption graph of the chromatographic column to the saffron extract in Example 3;
[0061] Fig.11 Schematic diagram of the multi-stage mass spectrometry fragmentation pathway of crocin I to IV standards under HCD collision energy in Example 4. DETAILED DESCRIPTION
[0062] The following examples further illustrate the targeted identification method of crocin compounds and the new crocin compounds of the present invention. The examples are only intended to illustrate the present invention, but not to limit the present invention.
[0063] Example 1
[0064] S1. Preparation of saffron extract and crocin standard:
[0065] Crocin I, Crocin II, Crocin III, and Crocin IV standard products were respectively prepared into 0.5 μg / mL standard solutions with 50% methanol to obtain crocin standard products.
[0066] Take the ground saffron stigma powder, add 50% methanol aqueous solution, and ultrasonically extract for 1 hour at a solid-liquid ratio of 20 mg:1 mL. After high-speed centrifugation, take the supernatant and dilute it with 50% methanol to 0.5 mg / mL to obtain saffron extract for liquid-to-mass analysis.
[0067] S2. Based on the core structure of crocin and the reported structural rules of crocin compounds, the possible substituent types of crocin are inferred, and a list of targeted parent ions is generated based on the possible substituents. The list of targeted parent ions includes [MH] -、[M+HCOO] - and [M+2HCOO] 2- Additive peak, crocin compound is generated by dehydration of crocetin with 1 to 6 glucose molecules or dehydration esterification with 1 to 2 methanol molecules;
[0068] In this embodiment, the target parent ion list is shown in Table 1:
[0069] Table 1 List of targeted precursor ions
[0070]
[0071]
[0072] S3, injecting saffron extract and crocin standard into a high performance liquid chromatography-mass spectrometer, and inputting the parent ions in the targeted parent ion list into the mass spectrometer as the target substance, so that the mass spectrometry acquisition data will give priority to the parent ions in the list to hit the secondary fragments, collect the secondary fragment ions in the parent ion list, and identify the target substance in a targeted manner. After testing, the ultraviolet absorption, parent ion mass-to-charge ratio, secondary to quaternary fragment ion mass-to-charge ratio and retention time of all crocin components in the saffron extract and the crocin standard are obtained respectively;
[0073] The specific testing conditions are as follows:
[0074] A Waters CORTECS UPLC C18 column (100 mm × 2.1 mm, 1.6 μm) was used, and the mobile phase was acetonitrile (A) and 0.1% formic acid water (B) system, with an elution gradient of: 0-10 min, 5-30% A; 10-15 min, 30-50% A; 15-18 min, 50-95% A; 18-21 min, 95% A; equilibrium time 3 min. The injection volume was 2 μL, the flow rate was 0.4 ml / min, and the DAD wavelengths were 260 nm, 330 nm, 440 nm, and 460 nm.
[0075] The Thermo Fisher Vanquish UHPLC-DAD system was connected to an Orbitrap ID-X Tribrid multistage mass spectrometer, with an electrospray ion source, negative ion scanning mode, capillary voltage, 3.0 kV; sheath gas, 40 arb; auxiliary gas, 10 arb; evaporation temperature, 300 °C; ion transfer tube temperature, 300 °C; primary scanning range, m / z 280-1500; primary resolution, 60000; secondary to quaternary resolution, 7500; collision energy, HCD / CID combined mode, MS 2 ,HCD stepped(10,20,40),MS 3 , HCD, 25, MS 4, CID,45.
[0076] Prioritize the ions in the parent ion list to generate secondary fragments, then select the three most abundant ions from the obtained secondary fragments to generate tertiary fragments again, and finally select the most abundant ions from the obtained tertiary fragments to generate quaternary fragments.
[0077] Figure 1 is the obtained UV spectrum (260nm), Figure 2 It is the mass spectrum (Extracted Ion Chromatogram, EIC diagram) obtained by extracting the targeted precursor ion list.
[0078] S4. By comparing the UV absorption, mass-to-charge ratio of the first to fourth fragment ions and retention time of all crocin components in the saffron extract with the UV absorption, parent ion mass-to-charge ratio, mass-to-charge ratio of the second to fourth fragment ions and retention time of crocin standards (crocin I, crocin II, crocin III, crocin IV), the known crocin compound types were identified;
[0079] Combining the multi-step cleavage rules and cleavage fragments of crocin as well as the corresponding ultraviolet absorption wavelengths, three new crocin compounds were identified from saffron, namely new compounds 1, 2 and 3.
[0080] Specific analysis and identification methods include:
[0081] By checking the corresponding retention time, multi-stage fragmentation information and ultraviolet absorption in the standard and saffron extract, the four chromatographic peaks with retention times of 8.80min, 9.68min, 10.70min and 12.40min corresponded to crocin I, crocin II, crocin III and crocin IV, respectively, and the known crocin compound types were identified. The multi-stage mass spectrometry fragmentation pathways of crocin I, crocin II, crocin III and crocin IV standards are shown in Figure 3 , Crocin I standard produced [M+HCOO]- ion (m / z 1021.38), MS 2 The main fragment ions produced were m / z 651.27 and m / z 975.37, corresponding to [MH-2glu] - and [MH] - , therefore, MS 2 The glycosidic bond will be broken. In addition to losing a molecule of formic acid, a neutral molecule of 324 is also lost, corresponding to a molecule of gentiobiose structure. In addition, MS 2 An ion of m / z 323.10 was also observed, corresponding to gentiobiose-H. -In the structure of crocin I, one side carboxyl group is directly connected to a molecule of gentiobiose to form a glycosidic bond; MS 3 The precursor ion m / z 651.27 was used for the third level, and the neutral molecule of 324 was lost, producing the aglycone fragment ion of m / z 327.16. The other side carboxyl group of crocin I formed an ester bond with gentiobiose; MS 4 The aglycone ion m / z 327.16 was cleaved to produce m / z 283.17 and m / z 239.18 fragment ions, corresponding to the loss of 1 to 2 CO2 molecules, and the carboxyl functional groups on both sides of the crocetin aglycone produced characteristic cleavage ions. Multi-stage cleavage proved that crocin I is a glycosidic bond formed by the carboxyl groups on both sides of crocetin with a molecule of gentiobiose, combined with the characteristic ultraviolet absorption wavelength (see Figure 4 ), all-trans (trans-) crocin has maximum absorption at around 440nm and 460nm, and no characteristic absorption at around 330nm. In addition to the maximum absorption at 440nm and 460nm, 13-cis (13-cis) crocin has maximum absorption at 330nm, proving that crocin I is in all-trans trans configuration, i.e., trans-4-(2G,2G). According to the above ideas, the structures of crocin II to IV are respectively trans-3-(1g,2G) (a trisaccharide with a glucose on one side and a gentiobiose structure on the other side), trans-2-(1g,1g) (a disaccharide with a glucose on each side) and trans-2G (a disaccharide with a gentiobiose on one side and no carboxyl substitution on the other side). Therefore, the structure of crocin can be jointly identified by multi-stage cleavage pathways and ultraviolet absorption.
[0082] According to the method of Example 1, three new compounds 1, 2 and 3 were identified by multi-step pyrolysis and UV absorption spectroscopy.
[0083] New compound 1(t R 8.01min, C 56 H 84 O 34 )'s multi-stage fragmentation mass spectrum and fragmentation pathways are shown in Figure 5 The first-level mass spectrometry of compound 1 extracted a chromatographic peak of m / z 695.24, which belongs to the double-charged peak of crocus-6 glycoside. 2 The m / z 651.26 fragment ion was generated, and a neutral molecule (648) with 4 glucose units was lost. At the same time, the m / z 647.20 fragment ion was generated, corresponding to the [4glu-H]- ion. Compound 1 may be a structure in which the carboxyl groups on both sides of the crocetin aglycone form glycosidic bonds with two glucoses at one end and with four glucoses at one side. 3The precursor ion further loses a neutral molecule with 2 glucose units (324) to produce aglycone ion m / z 327.16; MS 4 The aglycon ion was fragmented, losing one molecule of CO2, generating a fragment ion of m / z 283.17, with an MS value of 651.26. 3 The precursor ion, the fragmentation process is consistent with the standard crocin I standard. Compound 1 is speculated to be composed of crocetin connected to gentiobiose on one side and 4 glucose on the other side. Combined with the UV absorption spectrum of compound 1, the compound is trans-6-(2G, 4glu) configuration, and the 4 glucose units (4glu) exist in the configuration of linear tetrasaccharides and branched tetrasaccharides. With m / z 647.20 [4glu-H]- as the precursor ion, m / z 545.17 fragment ion is generated, and 102 neutral molecules are lost in neutral; m / z 383.12 is generated, and 262Da is lost in neutral, corresponding to the neutral loss of 1 glucose residue and 102 molecules (162+102); m / z 443.13 ion is generated, corresponding to the neutral loss of 204Da, and the fragmentation mode is shown in Figure 5 As shown in the figure, it is speculated that the tetrasaccharide belongs to a linear tetrasaccharide structure, namely [D-glucopyranosyl-(1β→6)-D-glucopyranosyl-(1β→6)-D-glucopyranosyl-(1β→6)-D-glucopyranose]. The literature [Ernst Pittenauer. PHYTOCHEMICAL ANALYSIS. 2019, 30(3), 346-356] compared the mass spectrometry fragmentation pathways of linear polysaccharides and branched polysaccharides. The linear polysaccharide 0,4 A3, 0,2 The A2 site cleavage produces characteristic neutral losses of 102Da and 204Da, which is consistent with the cleavage pathway of this experiment. Combined with the UV absorption spectrum ( Figure 8 ), with maximum absorption at around 420nm and 450nm, and no characteristic ultraviolet absorption at 330nm, it is judged to be an all-trans trans configuration. Therefore, the final structure of compound 1 is trans-6-(2G,4G), that is, trans-crocetin is connected to gentiobiose on one side and gentiotetraose (linear tetrasaccharide) on the other side. The final structure is as follows:
[0084]
[0085] New compound 2 (tR 9.05min, C 44 H 64 O 24 ), MS primary extraction m / z 533.19, m / z 1021.38, corresponding to [M+2HCOO] 2- Double charged peak and [M+HCOO] -Single charged peak, with m / z 1021.38 as MS 2 The precursor ion produced fragment ions of m / z 975.37, 813.31, 489.21, and 485.15, corresponding to [MH] - , [MH-glu] - , [MH-3glu] - , [3glu-H] - It is speculated that compound 2 is a trans / cis-4-(1g,3glu) structure, that is, crocin 4-glycoside, where the carboxyl group on one side of crocin acid forms a glycosidic bond with one glucose molecule, and the carboxyl group on the other side forms a glycosidic bond with three glucose molecules; in addition, MS 2 A series of carbohydrate fragment ions were also generated, which were presumably generated by the cleavage of the triglucose residue m / z 485.15, 383.12 (neutral loss 102 Da), 341.11 (neutral loss 144 Da), 323.10, 221.06, 179.05, and 161.04. The specific mass spectrometry fragmentation process is shown in Figure 6 The fragment ions obtained in the experiment are consistent with the cleavage pathway of the linear trisaccharide (gentiotriose, [D-glucopyranosyl-(1β→6)-D-glucopyranosyl-(1β→6)-D-glucopyranose]). Therefore, combined with the UV absorption spectrum ( Figure 8 ), it is speculated that compound 2 is trans-4-(1g,3G) with the following structure:
[0086]
[0087] New compound 3(t R 14.98min, C 33 H 46 O 14 ), m / z 711.28 is [M+HCOO] of compound 3 - Added peak, mass spectrum fragmentation diagram see Figure 7 , with [M+HCOO] - is the precursor ion, MS 2 Mainly produces 665.30[MH] - ,341.19[MH-2glu] - , and m / z 323.10 [Gentiobiose-H] - ion, it is speculated that one side of the carboxyl group dehydrates with a gentiobiose molecule to form a glycosidic bond, with m / z 341.19 [MH-2glu] - For MS 3The precursor ion produced m / z 297.18 ion, corresponding to the neutral loss of 1 molecule of CO2, produced m / z 265.16 fragment ion, corresponding to the loss of 1 molecule of CO2 and 1 molecule of CH3OH, and produced m / z 237.16, corresponding to the loss of 1 molecule of CO2, 1 molecule of CH3OH and 1 molecule of CO. It is speculated that compound 3 is a glycosidic bond formed by the dehydration of the carboxyl group on one side of crocetin with gentiobiose, and a monomethyl ester bond formed by the dehydration of the carboxyl group on the other side with methanol. The UV absorption has maximum absorption at 440nm and 460nm, and no maximum absorption at 330nm (UV absorption spectrum see Figure 8 ), it is speculated to be the all-trans configuration, that is, trans-crocin bioside monomethyl ester trans-2G methyl ester, the structure of which is as follows:
[0088]
[0089] Example 2
[0090] The difference from Example 1 is that in step S3, an ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm) chromatographic column is used, the mobile phase is an acetonitrile (A) and 0.1% formic acid water (B) system, and the elution gradient is: 0-10 min, 5-30% A; 10-13 min, 30-50% A; 13-15 min, 50-90% A.
[0091] UV absorption Fig. 9 .
[0092] Example 3
[0093] The difference from Example 2 is that in step S1, 100% methanol aqueous solution is used as the extraction solvent for saffron powder.
[0094] UV absorption diagram Fig.10 .
[0095] Example 4
[0096] The difference from Example 1 is that in step S3, the collision mode is HCD (high energy induced cracking) and the collision energy is MS 2 HCD stepped(10,20,40),MS 3 HCD25,MS 4 HCD45; see the multi-step cleavage pathway of crocin I to IV for details. Fig.11 .
[0097] The above are only examples of the features of the present invention and do not constitute any limitation on the protection scope of the present invention. Any technical solution formed by equivalent exchange or equivalent replacement falls within the protection scope of the present invention.
Claims
1. A targeted identification method for crocin compounds, characterized in that: The following steps are involved: S1. preparing saffron extract and crocin standard; S2. Based on the core structure of crocin and the reported structural rules of crocin compounds, the possible substituent types of crocin are inferred, and a list of targeted parent ions is generated based on the possible substituents. The list of targeted parent ions includes [MH] - 、[M+HCOO] - and [M+2HCOO] 2- Additive peak, crocin compound is generated by dehydration of crocetin with 1 to 6 glucose molecules or dehydration esterification with 1 to 2 methanol molecules; S3, injecting the saffron extract and the crocin standard into a high performance liquid chromatography-mass spectrometer, and inputting the ions in the target parent ion list into the mass spectrometer as target substances, and obtaining the ultraviolet absorption, parent ion mass-to-charge ratio, secondary to quaternary fragment ion mass-to-charge ratio and retention time of all crocin components in the saffron extract and the crocin standard respectively; S4. Identify known crocin compound types by comparing the UV absorbance, mass-to-charge ratio of primary to quaternary fragment ions and retention time of all crocin components in the saffron extract with the UV absorbance, parent ion mass-to-charge ratio, mass-to-charge ratio of secondary to quaternary fragment ions and retention time of crocin standards; Combining the multi-stage cleavage rules of crocin, the cleavage fragments and the corresponding UV absorption wavelengths, three new crocin compounds were identified from saffron extract. In step S3, the ultra high performance liquid chromatography conditions include: Chromatographic column: C18 column; Mobile phase: Phase A is acetonitrile, phase B is 0.1%-0.5% formic acid water or 0.1%-0.5% acetic acid water; Elution gradient: 0-10min, 5-30% A; 10-15min, 30-50% A; 15-18min, 50-95%A; 18-21min, 95%A; Column temperature 20-40°C; The detection wavelength is 190-600nm; In step S3, the C18 chromatographic column is selected from one of CORTECS UPLC C18, ACQUITY UPLC BEH C18 and Zorbax SB-C18; In step S3, the mass spectrometry conditions include: ESI ion source: negative ion mode; Evaporation temperature: 250-350℃; Ion transfer tube temperature: 250-350℃; Auxiliary gas: 30-40Arb; Primary parameters: MS scanning range, m / z 100-1500; Input the ions in the target precursor ion list into the mass spectrometer as the target substances, give priority to the precursor ions in the target precursor ion list for secondary fragmentation, then select the three most abundant ions from the obtained secondary fragments to perform tertiary fragmentation again, and finally select the most abundant ions from the obtained tertiary fragments to perform quaternary fragmentation; Multi-level parameters: MS2~MS4 scanning range, m / z 50-1500; HCD or CID collision mode; In step S1, the preparation method of saffron extract includes: taking the ground saffron stigma powder, adding 50-100% methanol aqueous solution or 30-100% ethanol aqueous solution, ultrasonically extracting for 0.5-1.5 hours at a solid-liquid ratio of 15-25 mg:1 mL, centrifuging and taking the supernatant and diluting it to 0.5 mg / mL with 50% methanol; the preparation method of crocin standard includes: taking crocin I, crocin II, crocin III, and crocin IV standard products respectively, and configuring them into a 0.5 μg / mL standard solution with 50% methanol; In step S2, the targeted precursor ion list is specifically: The structural formulas of the three new crocin compounds are: Crocin hexaglycoside: Crocin Tetrasaccharoside: Monomethyl esterified crocin bioside: