Method for detecting chemical components in Mailuoning oral liquid based on HPLC-QTOF-MS / MS technology

Abstract

The invention discloses a method for detecting chemical components in a Mailuoning oral liquid based on an HPLC-QTOF-MS / MS. The method is characterized by comprising the following steps: taking a Mailuoning oral liquid test solution and a reference substance solution, carrying out high performance liquid chromatography separation, respectively collecting data in a positive ion mode and a negative ion mode by using a time-of-flight mass spectrometry, and determining the content of chemical components in the Mailuoning oral liquid. And comparing mass spectrums and fragment information of chromatographic peaks with the same retention time in the reference substance and the test sample to identify the chemical components in the Mailuoning oral liquid. According to the method, the HPLC-QTOF-MS / MS method is adopted for qualitative detection of multiple types of chemical components in the Mailuoning oral liquid for the first time, and the method has the advantages of being efficient, simple, convenient, rapid, accurate and comprehensive. 54 chemical components are identified by adopting the method disclosed by the invention, and reference is provided for further researching the pharmacodynamic material basis and comprehensive quality control of the Mailuoning oral liquid.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medical component analysis, and particularly relates to a method for detecting chemical components in Mailuoning oral liquid based on HPLC-QTOF-MS / MS technology. BACKGROUND

[0002] Mailuoning oral liquid has the effects of clearing heat and nourishing yin and promoting blood circulation to remove blood stasis. The prescription is composed of five Chinese medicinal materials, including Radix Cyathulae, Radix Scrophulariae, Dendrobium, Flos Lonicerae Japonicae and Flos Lonicerae, and is commonly used for treating thromboangiitis obliterans, venous thrombosis, arteriosclerotic occlusion, cerebral thrombosis and its sequelae.

[0003] Mailuoning oral liquid is widely used, but the pharmaceutical research is relatively less, especially the research on the chemical components is less. SUMMARY

[0004] The purpose of the application is to provide a method for detecting chemical components in Mailuoning oral liquid based on HPLC-QTOF-MS / MS technology, to qualitatively analyze the chemical components in Mailuoning oral liquid, and to provide a reference for further studying the pharmacodynamic material basis and comprehensive quality control of Mailuoning oral liquid.

[0005] In order to achieve the above-mentioned purpose of the application, the technical scheme provided by the application is as follows:

[0006] A method for detecting chemical components in Mailuoning oral liquid based on HPLC-QTOF-MS / MS technology, comprising: taking Mailuoning oral liquid test solution and control solution, separating by high performance liquid chromatography, collecting data in positive ion and negative ion modes by time-of-flight mass spectrometry, comparing the mass spectra and fragment information of the chromatographic peaks with the same retention time in the control and test samples, and identifying the chemical components in Mailuoning oral liquid in combination with literature references.

[0007] The data are primary and secondary mass spectrometry fragments and retention time.

[0008] The Mailuoning oral liquid test solution is a filtrate obtained by diluting Mailuoning oral liquid with water and filtering through a filter membrane.

[0009] Preferably, the Mailuoning oral liquid test solution is a filtrate obtained by diluting Mailuoning oral liquid with water to 10 times the volume of Mailuoning oral liquid, and then filtering through a 0.22 mu water system filter membrane.

[0010] The preparation method of the control solution is as follows: precisely weigh at least one of gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harbarin, neochlorogenic acid, protocatechualdehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-hydroxycinnamic acid, methyl chlorogenic acid, ferulic acid, forsythoside, binghao lactone, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, jacoboside, methyl caffeate, hyperoside, rutin, β-ecdysterone, 4,5-dicaffeoylquinic acid, angloin C, methyl p-hydroxycinnamate, cinnamic acid, ethyl caffeate, luteolin, harbarin, baicalin, macrostides B, macrostides A, notoginsenoside B standard substance, and then dissolve in methanol to constant volume.

[0011] Preferably, the control solution comprises mixed standard 1 and mixed standard 2; the mixed standard 1 is a mixed methanol solution of gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harpagide, neochlorogenic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-hydroxycinnamic acid, methylchlorogenate, ferulic acid, forsythoside, scabrosid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, methyl caffeate, hyperoside, rutin, β-ecdysterone, 4,5-dicaffeoylquinic acid, angloides C, methyl p-hydroxycinnamate, cinnamic acid, ethyl caffeate, luteolin, harpagide, baicalin, and bartsioside B; in the mixed standard 1, the concentrations of gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harpagide, neochlorogenic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-hydroxycinnamic acid, methylchlorogenate, ferulic acid, forsythoside, scabrosid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, methyl caffeate, hyperoside, rutin, β-ecdysterone, 4,5-dicaffeoylquinic acid, angloides C, methyl p-hydroxycinnamate, cinnamic acid, ethyl caffeate, luteolin, harpagide, baicalin, and bartsioside B are 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 110-130 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 120-140 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 120-140 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 140-160 μg / mL, 80-100 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 120-140 μg / mL, 80-100 μg / mL, 100-120 μg / mL, 100-120 μg / mL, respectively.The mixed marker 2 is a mixed methanol solution of luteoloside and sibirienoside A, and in the mixed marker 2, the concentrations of luteoloside and sibirienoside A are 100-120 μg / mL and 100-120 μg / mL respectively.

[0012] Preferably, in the mixed marker 1, the concentrations of gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harpagide, neochlorogenic acid, protocatechualdehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-coumaric acid, methylchlorogenate, ferulic acid, saikosaponin a, scabrosin, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, methyl caffeate, hyperoside, rutin, β-ecdysterone, 4,5-dicaffeoylquinic acid, angloin C, methyl p-hydroxycinnamate, cinnamic acid, ethyl caffeate, luteolin, harpagide, baicalin, sibirienoside B, and asperosaponin B are 110 μg / mL, 115 μg / mL, 112 μg / mL, 109 μg / mL, 126 μg / mL, 116 μg / mL, 108 μg / mL, 131 μg / mL, 134 μg / mL, 115 μg / mL, 110 μg / mL, 112 μg / mL, 123 μg / mL, 121 μg / mL, 107 μg / mL, 124 μg / mL, 117 μg / mL, 105 μg / mL, 146 μg / mL, 91 μg / mL, 104 μg / mL, 109 μg / mL, 113 μg / mL, 115 μg / mL, 103 μg / mL, 113 μg / mL, 106 μg / mL, 107 μg / mL, 112 μg / mL, 108 μg / mL, 114 μg / mL, 116 μg / mL, 122 μg / mL, 125 μg / mL, 98 μg / mL, 108 μg / mL, and 101 μg / mL respectively; and in the mixed marker 2, the concentrations of luteoloside and sibirienoside A are 104 μg / mL and 103 μg / mL respectively.

[0013] The high performance liquid chromatography conditions are as follows: the chromatographic column is a reversed-phase Plus C 18Chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); 0.1%~0.5% formic acid water as mobile phase A, methanol as mobile phase B; gradient elution program: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate 0.6~1.0 mL / min; column temperature 25~35 ℃; injection volume 10 μL.

[0014] Preferably, the high performance chromatography condition is that the chromatographic column is a reversed-phase Plus C 18 Chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); 0.1% formic acid water as mobile phase A, methanol as mobile phase B; gradient elution program: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate 1.0 mL / min; column temperature 30 ℃; injection volume 10 μL.

[0015] The mass spectrometry condition is that the ion source is an ESI source; positive / negative ion mode is scanned respectively; atomizing gas flow rate: 9.0 L / min; atomizing gas pressure: 35 psi; ion source temperature: 325 ℃; spray voltage: +3500 V / -4000 V; collision voltage: 120 V; cone hole voltage: 65 V; collision energy: 10 V / 30 V / 50 V; scanning range: m / z 100~2000.

[0016] The beneficial effects of the present application are:

[0017] The present application firstly adopts HPLC-QTOF-MS / MS method to qualitatively detect multiple types of chemical components in Mailuoning oral liquid, and has the characteristics of high efficiency, simplicity, rapidness, accuracy and comprehensiveness. 54 kinds of chemical components are identified by the method, which provides a reference for further studying the efficacy material basis and comprehensive quality control of Mailuoning oral liquid. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The chromatogram of Mailuoning oral liquid when different chromatographic columns are used.

[0019] Figure 2 The chromatogram of Mailuoning oral liquid when different mobile phase compositions are used.

[0020] Figure 3The chromatogram of Mailuoning oral liquid at different formic acid concentrations.

[0021] Figure 4 The chromatogram of Mailuoning oral liquid at different column temperatures.

[0022] Figure 5 The chromatogram of Mailuoning oral liquid at different flow rates.

[0023] Figure 6 The total ion flow chart of Mailuoning oral liquid under positive / negative ions.

[0024] Figure 7 The total ion flow chart of control solution HB1 under positive / negative ions.

[0025] Figure 8 The total ion flow chart of control solution HB2 under positive / negative ions.

[0026] Figure 9 The mass spectrum information chart and fragmentation pathway chart of hypaphorina cryptomeriana in Mailuoning oral liquid; wherein, A: MS chart; B: MS / MS chart; C: fragmentation pathway chart. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be described in detail below in combination with examples. It should be particularly pointed out that the technical solutions of the present application have been described by preferred examples, and those skilled in the art can refer to the present application to appropriately improve process parameters for implementation without departing from the content, spirit and scope of the present application. All similar replacements and modifications are obvious to those skilled in the art, and they are considered to be included in the present application.

[0028] Instrument: Agilent 1260 HPLC-6530 QTOF liquid chromatograph-mass spectrometer; one hundredth of an electronic scale (METTLER TOLEDO).

[0029] Reagents: Mailuoning oral liquid (batch number: 191201), Nanjing Jinling Pharmaceutical Factory of Jinling Pharmaceutical Co., Ltd.; gallic acid (CAS: 149-91-7), onjisaponin (CAS: 479-98-1), 5-hydroxymethylfurfural (CAS: 67-47-0), 1-caffeoylquinic acid (CAS: 1241-87-8), protocatechuic acid (CAS: 99-50-3), harpagide (CAS: 6926-08-5), neochlorogenic acid (CAS: 906-33-2), protocatechualdehyde (CAS: 139-85-5), p-hydroxybenzoic acid (CAS: 99-96-7), dihydrocaffeic acid (CAS: 1078-61-1), chlorogenic acid (CAS: 327-97-9), cryptochlorogenic acid (CAS: 905-99-7), vanillic acid (CAS: 121-34-6), caffeic acid (CAS: 331-39-5), gardenoside (CAS: 24512-62-7), p-coumaric acid (CAS: 7400-08-0), methyl chlorogenate (CAS: 123483-19-2), ferulic acid (CAS: 1135-24-6), scabioside (CAS: 51938-32-0), binfarolide (CAS: 120-08-1), 3,4-dicaffeoylquinic acid (CAS: 14534-61-3), 3,5-dicaffeoylquinic acid (CAS: 2450-53-5), 1,5-dicaffeoylquinic acid (CAS: 19870-46-3), jacoboside (CAS: 5373-11-5), methyl caffeate (CAS: 3843-74-1), hyperoside (CAS: 482-36-0), rutin (CAS: 153-18-4), β-ecdysterone (CAS: 5289-74-7), 4,5-dicaffeoylquinic acid (CAS: 32451-88-0), angloideside C (CAS: 115909-22-3), methyl p-hydroxycinnamate (CAS: 19367-38-5), cinnamic acid (CAS: 621-82-9), ethyl caffeate (CAS: 102-37-4), luteolin (CAS: 491-70-3), harpagide (CAS: 19210-12-9), baohuoside A (CAS: 95041-90-0), campneoside Ⅱ (CAS: 136849-88-2), campneoside Ⅰ (CAS: 140360-29-8), and asperosaponin Ⅱ (CAS: 33289-85-9) were purchased from Chengdu Desite Biological Technology Co., Ltd. and used as the reference substances (purity ≥98%). Methanol (LC-MS grade) was purchased from Merck, formic acid (LC-MS grade) was purchased from Thermo, and ultrapure water was self-made.

[0030] Example 1

[0031] Preparation of reference solution:

[0032] Take gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harbarin, neochlorogenic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-coumaric acid, methylchlorogenate, ferulic acid, forsythoside, blumenol, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, methyl caffeate, hyperoside, rutin, β-ecdysterone, 4,5-dicaffeoylquinic acid, angloin C, methyl p-coumarate, cinnamic acid, ethyl caffeate, luteolin, harbarin, baicalin, macrostides, and sibiricin, accurately weigh an appropriate amount of each, dissolve in methanol and dilute to a concentration of 110 μg / mL, 115 μg / mL, 112 μg / mL, 109 μg / mL, 126 μg / mL, 116 μg / mL, 108 μg / mL, 131 μg / mL, 134 μg / mL, 115 μg / mL, 110 μg / mL, 112 μg / mL, 123 μg / mL, 121 μg / mL, 107 μg / mL, 124 μg / mL, 117 μg / mL, 105 μg / mL, 146 μg / mL, 91 μg / mL, 104 μg / mL, 109 μg / mL, 113 μg / mL, 115 μg / mL, 103 μg / mL, 113 μg / mL, 106 μg / mL, 107 μg / mL, 112 μg / mL, 108 μg / mL, 114 μg / mL, 116 μg / mL, 122 μg / mL, 125 μg / mL, 98 μg / mL, 108 μg / mL, 101 μg / mL, respectively, to obtain a mixed reference solution (HB1).

[0033] Take luteoloside and macrostides A reference substances, accurately weigh an appropriate amount of each, dissolve in methanol and dilute to a concentration of 104 μg / mL, 103 μg / mL, respectively, to obtain a mixed reference solution (HB2).

[0034] Preparation of test solution of Mailuoning oral liquid: accurately take 1 mL of Mailuoning oral liquid, place it in a 10 mL volumetric flask, add water to the mark, shake well, filter through a 0.22 μm water filter membrane, and take the filtrate to obtain the test solution.

[0035] Optimization of chromatographic conditions:

[0036] (1) Selection of chromatographic column

[0037] In order to obtain better peak conditions and separation effect, the inventors compared different models of chromatographic columns from different manufacturers, including Agilent ZORBAX Extend-C18 Agilent ZORBAX Extend-C Plus C 18 Agilent ZORBAX Extend-C C 18 Agilent ZORBAX Extend-C

[0038] High performance liquid chromatography conditions: 0.1% formic acid water as mobile phase A, methanol as mobile phase B; gradient elution program: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate is 1.0 mL / min; column temperature is 30°C; using Jingluoning oral liquid sample solution for injection, the injection amount is 10 μL. Ensure that the above chromatographic conditions remain unchanged, investigate different chromatographic columns (Agilent ZORBAX Extend-C 18 Agilent ZORBAX Extend-C Plus C 18 Agilent ZORBAX Extend-C C 18 Agilent ZORBAX Extend-C

[0039] The results are shown in Figure 1 It can be seen that: Agilent ZORBAX Extend-C 18 The separation effect of the chromatographic column is not good, and some chromatographic peaks cannot reach baseline separation; Waters C 18 The number of peaks of the chromatographic column is less and the peak shape is poor; and Plus C 18 The separation effect of the chromatographic column is the best in the case of more peak numbers, so the Plus C 18 chromatographic column is selected.

[0040] (2) Mobile phase screening

[0041] The composition of the mobile phase also greatly affects the chromatographic behavior of the sample. Different mobile phases have different elution abilities, so the inventors further investigated the effects of organic chromatographic solvents (methanol, acetonitrile) and water phase additives (formic acid, acetic acid, phosphoric acid, trifluoroacetic acid) on the separation of the sample.

[0042] High performance liquid chromatography conditions: Plus C 18Chromatographic column (4.6 x 250 mm, 3.5 μm); gradient elution procedure: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate 1.0 mL / min; column temperature 30°C; using test sample solution for injection, injection volume 10 μL.

[0043] While ensuring the above chromatographic conditions remain unchanged, investigate different mobile phase compositions: water as mobile phase A, acetonitrile as mobile phase B; water as mobile phase A, methanol as mobile phase B; 0.1% acetic acid water as mobile phase A, methanol as mobile phase B; 0.1% trifluoroacetic acid water as mobile phase A, methanol as mobile phase B; 0.1% formic acid water as mobile phase A, methanol as mobile phase B; 0.1% phosphoric acid water as mobile phase A, methanol as mobile phase B.

[0044] Results are shown in Table 1. Figure 2 It can be seen that: when methanol is used as the organic phase, the number of chromatographic peaks is greater and the baseline is flatter, which is superior to acetonitrile; in addition, when pure water is used as the water phase, the number of chromatographic peaks is significantly reduced; and for the same concentrations of formic acid, acetic acid, phosphoric acid and trifluoroacetic acid, under the condition of similar number of peaks, the chromatographic peak separation effect of methanol-formic acid is better.

[0045] (3) Formic acid concentration screening

[0046] Further investigate the influence of different formic acid concentrations (0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%) on the separation of Mailuoning Oral Liquid.

[0047] High performance liquid chromatography conditions: Plus C 18 Chromatographic column (4.6 x 250 mm, 3.5 μm); gradient elution procedure: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate 1.0 mL / min; column temperature 30°C; using test sample solution for injection, injection volume 10 μL. While ensuring the above chromatographic conditions remain unchanged, investigate the influence of different concentrations of formic acid water (0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5% formic acid water) as mobile phase A on the separation of chemical components in Mailuoning Oral Liquid when methanol is used as the organic phase (mobile phase B).

[0048] Results are shown in Table 1. Figure 3It can be seen that: different concentrations of formic acid have good peak and separation effects on the oral liquid of Mailuoning; however, when 0.01%, 0.02% and 0.05% formic acid is used, some chromatographic peaks cannot reach baseline separation, while when 0.1%, 0.2% and 0.5% formic acid is used, there is no obvious difference in chromatographic separation, therefore, 0.1%-0.5% formic acid is selected, and 0.1% formic acid is preferably used as the water phase (mobile phase A).

[0049] (4) Column temperature screening

[0050] The column temperature can not only affect the running time of the sample, but also affect the separation of the sample. The inventors investigated the influence of different column temperatures (25, 28, 30, 33 and 35°C) on the separation of the oral liquid of Mailuoning.

[0051] High performance liquid chromatography conditions: referring to the high performance liquid chromatography conditions of the formic acid concentration screening, only adjusting the mobile phase A to 0.1% formic acid, and the column temperature to 25, 28, 30, 33 and 35°C respectively.

[0052] The results are shown in Figure 4 It can be seen that: different column temperatures have little influence on the oral liquid of Mailuoning, and can achieve good separation effects, and the column temperature of 30°C is preferably selected.

[0053] (5) Mobile phase flow rate screening

[0054] Different mobile phase flow rates can affect the peak time and separation degree of the chromatographic peaks, and the inventors investigated the influence of different flow rates (0.6, 0.8, 1.0, 1.2 and 1.4 mL / min) on the separation of the oral liquid of Mailuoning, so as to optimize the better chromatographic conditions.

[0055] High performance liquid chromatography conditions: referring to the high performance liquid chromatography conditions of the formic acid concentration screening, only adjusting the mobile phase A to 0.1% formic acid, and the flow rate to 0.6, 0.8, 1.0, 1.2 and 1.4 mL / min respectively.

[0056] The results are shown in Figure 5 It can be seen that: when the flow rate is 1.2 and 1.4 mL / min, the separation effects of some chromatographic peaks are affected, and baseline separation cannot be achieved; when the flow rate is 0.6-1.0 mL / min, the chromatographic peaks achieve baseline separation, but when the flow rate is lower than 1.0 mL / min, the peak time of the chromatographic peaks is prolonged, therefore, the flow rate of 1.0 mL / min is preferably selected.

[0057] In summary, the determination method of the application is determined as follows: precisely taking 10 μL of the test sample solution and the control sample solution respectively, and injecting into the liquid chromatograph-mass spectrometer, and the high performance liquid chromatography conditions are as follows: the chromatographic column is a reversed-phase Plus C 18Chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); 0.1%~0.5% formic acid water as mobile phase A, methanol as mobile phase B; gradient elution program: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate 0.6~1.0 mL / min; column temperature 25~35 ℃; injection volume 10 μL.

[0058] The most preferred high performance liquid chromatography conditions are: the chromatographic column is a reversed-phase Plus C 18 Chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); 0.1% formic acid water as mobile phase A, methanol as mobile phase B; gradient elution program: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate 1.0 mL / min; column temperature 30 ℃; injection volume 10 μL.

[0059] Example 2

[0060] Prepare the mixed standard 1 (HB1), mixed standard 2 (HB2) and Maluoning oral liquid test solution according to Example 1, precisely take 10 μL of the test solution and the control solution HB1, HB2 respectively, inject into the liquid chromatograph-mass spectrometer, and the high performance liquid chromatography conditions are: the chromatographic column is a reversed-phase Plus C 18 Chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); 0.1% formic acid water as mobile phase A, methanol as mobile phase B; gradient elution program: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; flow rate 1.0 mL / min; column temperature 30 ℃; injection volume 10 μL. The mass spectrometry conditions are: the ion source is ESI source; positive / negative ion mode scanning respectively; atomizing gas flow rate: 9.0 L / min; atomizing gas pressure: 35 psi; ion source temperature: 325 ℃; spray voltage: +3500 V / -4000 V; collision voltage: 120 V; cone hole voltage: 65 V; collision energy: 10 V / 30 V / 50 V; scanning range: m / z 100~2000. Record the total ion current chromatogram.

[0061] Figure 6 Total ion chromatogram of the test solution of Mailuoning oral liquid under positive / negative ion conditions; Figure 7 This is the total ion chromatogram of the reference solution HB1 under positive / negative ion conditions; Figure 8 This is the total ion chromatogram of the reference solution under HB2 positive / negative ion conditions.

[0062] First-order and second-order mass spectrometry fragments and retention times were collected. Qualitative analysis of the chromatographic peaks was performed using the Agilent MassHunter Qualitative Analysis B.08.00 software integrated into the Agilent 1260HPLC-6530QTOF LC-MS system. Mass spectra and fragment information of peaks with the same retention times in the reference and test samples were compared, and literature was consulted. A total of 54 components were detected in the Mailuoning oral liquid, including 22 phenolic acids, 12 iridoid glycosides, 6 flavonoids, 5 triterpenoid saponins, 4 phenylethanol glycosides, and 5 other compounds. Specific results are shown in Table 1. Taking compound 15 as an example, based on its first-order mass spectrometry information (… Figure 9 A) The molecular ion peak [MH] was obtained. - Given m / z 353.0885, calculate its molecular formula as C. 16 H 18 O9, and then from its secondary mass spectrum, fragment ions with m / z 191.0561 ([MH-caffeoyl)) can be observed. - ), m / z 179.0353 ([MH-quinic] - ) and m / z 135.0451([MH-quinic-CO2] - ()( Figure 9 B) is derived from the loss of caffeoyl group, quinic acid, and neutral CO2 molecule from the primary ion. Finally, by comparing its retention time with the reference standard, it was accurately identified as cryptochlorogenic acid. Its cleavage pathway is shown in the figure below. Figure 9 C). Other compounds were also identified by comparing information such as primary and secondary mass spectrometry fragmentation and retention times with literature.

[0063]

[0064]

[0065]

Claims

1. A method for detecting chemical components in Mailuoning oral liquid based on HPLC-QTOF-MS / MS technology, characterized in that: The method comprises the following steps: The method comprises the following steps: taking the test sample solution and the control sample solution of the Mailuoning oral liquid, separating them by high performance liquid chromatography, collecting data in positive ion and negative ion modes respectively by time-of-flight mass spectrometry, comparing the mass spectra and fragment information of the chromatographic peaks with the same retention time in the control sample and the test sample, and identifying the chemical components in the Mailuoning oral liquid by combining with literature references. 2.The method for detecting chemical components in Mailuoning Oral Liquid based on HPLC-QTOF-MS / MS technology according to claim 1, characterized in that: The test sample solution of the Mailuoning oral liquid is obtained by diluting the Mailuoning oral liquid with water and filtering the dilution through a filter membrane. 3.The method for detecting chemical components in Mailuoning Oral Liquid based on HPLC-QTOF-MS / MS technology according to claim 2, characterized in that: The test sample solution of the Mailuoning oral liquid is obtained by diluting the Mailuoning oral liquid with water to 10 times the volume of the Mailuoning oral liquid, and then filtering the dilution through a 0.22 μm water filter membrane. 4.The method for detecting chemical components in Mailuoning Oral Liquid based on HPLC-QTOF-MS / MS technology according to claim 1, characterized in that: The control sample solution is prepared by accurately weighing at least one of the following standard substances: gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harbarin, neochlorogenic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-hydroxycinnamic acid, methyl chlorogenic acid, ferulic acid, scrophularinoside, bingmayine lactone, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, jacoboside, methyl caffeic acid, hyperoside, rutin, beta-ecdysterone, 4,5-dicaffeoylquinic acid, angloin C, methyl p-hydroxycinnamate, cinnamic acid, ethyl caffeic acid, luteolin, harbarin, baicalin, honeysuckle saponin B, honeysuckle saponin A, and notoginseng saponin B, and then dissolving them in methanol.

5. The method for detecting chemical components in the oral liquid of Mailuoning according to claim 1 or 4, characterized in that: The control solution comprises mixed standard 1 and mixed standard 2; the mixed standard 1 is a mixed methanol solution of gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harpagide, neochlorogenic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-hydroxycinnamic acid, methylchlorogenate, ferulic acid, forsythoside, scabrosin, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, methyl caffeate, hyperoside, rutin, beta-ecdysterone, 4,5-dicaffeoylquinic acid, angloides C, methyl p-hydroxycinnamate, cinnamic acid, ethyl caffeate, luteolin, harpagide, baicalin, and bartsioside B; in the mixed standard 1, the concentrations of gallic acid, aucubin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harpagide, neochlorogenic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-hydroxycinnamic acid, methylchlorogenate, ferulic acid, forsythoside, scabrosin, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, methyl caffeate, hyperoside, rutin, beta-ecdysterone, 4,5-dicaffeoylquinic acid, angloides C, methyl p-hydroxycinnamate, cinnamic acid, ethyl caffeate, luteolin, harpagide, baicalin, and bartsioside B are 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 110-130 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 120-140 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 120-140 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 140-160 μg / mL, 80-100 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 120-140 μg / mL, 120-140 μg / mL, 80-100 μg / mL, 100-120 μg / mL, 100-120 μg / mL, respectively.The mixed marker 2 is a mixture of methanol solution of luteoloside and sibirienoside A, and the concentration of luteoloside and sibirienoside A in the mixed marker 2 is 100-120 μg / mL and 100-120 μg / mL, respectively. 6.The method for detecting chemical components in Mailuoning Oral Liquid based on HPLC-QTOF-MS / MS technology according to claim 5, characterized in that: The concentration of gallic acid, onjisaponin, 5-hydroxymethylfurfural, 1-caffeoylquinic acid, protocatechuic acid, harpagide, neochlorogenic acid, protocatechualdehyde, p-hydroxybenzoic acid, dihydrocaffeic acid, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, gardenoside, p-coumaric acid, methylchlorogenate, ferulic acid, forsythoside, bingmayonglactone, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, methyl caffeate, hyperoside, rutin, β-ecdysterone, 4,5-dicaffeoylquinic acid, angloideside C, methyl p-coumarate, cinnamic acid, ethyl caffeate, luteolin, harpagide, baicalin, macrocyclic lactone, acteoside, chloroethyl, the concentration of the mixed marker 1 is respectively 110 μg / mL, 115 μg / mL, 112 μg / mL, 109 μg / mL, 126 μg / mL, 116 μg / mL, 108 μg / mL, 131 μg / mL, 134 μg / mL, 115 μg / mL, 110 μg / mL, 112 μg / mL, 123 μg / mL, 121 μg / mL, 107 μg / mL, 124 μg / mL, 117 μg / mL, 105 μg / mL, 146 μg / mL, 91 μg / mL, 104 μg / mL, 109 μg / mL, 113 μg / mL, 115 μg / mL, 103 μg / mL, 113 μg / mL, 106 μg / mL, 107 μg / mL, 112 μg / mL, 108 μg / mL, 114 μg / mL, 116 μg / mL, 122 μg / mL, 125 μg / mL, 98 μg / mL, 108 μg / mL, 101 μg / mL; the concentration of the mixed marker 2 is respectively 104 μg / mL and 103 μg / mL. 7.The method for detecting chemical components in Mailuoning Oral Liquid based on HPLC-QTOF-MS / MS technology according to claim 1, characterized in that: The high performance liquid chromatography condition is as follows: the chromatographic column is reversed phase Plus C 18 The chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 3.5 μm; 0.1%-0.5% formic acid water is used as mobile phase A, and methanol is used as mobile phase B; the gradient elution procedure is as follows: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; the flow rate is 0.6-1.0 mL / min; the column temperature is 25-35 ℃; and the injection volume is 10 μL. 8.The method for detecting chemical components in Mailuoning Oral Liquid based on HPLC-QTOF-MS / MS technology according to claim 1, characterized in that: The high performance chromatography condition is as follows: the chromatographic column is reversed phase Plus C 18 The chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 3.5 μm; 0.1% formic acid water is used as mobile phase A, and methanol is used as mobile phase B; the gradient elution program is as follows: 0-10 min, 3%-20% B; 10-45 min, 20%-55% B; 45-65 min, 55%-100% B; 65-80 min, 100% B; 80-85 min, 100%-3% B; 85-90 min, 3% B; the flow rate is 1.0 mL / min; the column temperature is 30 °C; and the injection amount is 10 μL.

9. The method for detecting chemical components in Mailuoning oral liquid based on HPLC-QTOF-MS / MS technology according to claim 1, wherein the mass spectrometry conditions are: the ion source is ESI source; positive / negative ion mode is scanned respectively; the atomization gas flow rate is 9.0 L / min; the atomization gas pressure is 35 psi; the ion source temperature is 325℃; the spray voltage is +3500V / -4000V; the collision voltage is 120V; the cone hole voltage is 65V; the collision energy is 10V / 30V / 50V; the scanning range is m / z 100-2000. ​

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