Method for determining chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology

Abstract

The invention discloses a method for determining chemical components in Mailuoning oral liquid based on a UPLC-QqQ-MS / MS technology. The method comprises the following steps: preparing a reference substance solution; preparing a test solution; establishing a linear regression equation; and detection of the test solution: precisely sucking the test solution, injecting the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer for detection, substituting the peak area into the established linear regression equation, calculating to obtain the content of each chemical component in the test solution, and converting to obtain the content of the chemical component in the Mailuoning oral liquid. According to the method, the UPLC-QqQ-MS / MS method is adopted for the first time, meanwhile, the 30 chemical components in the Mailuoning oral liquid are quantitatively determined, the method has the advantages of being simple, rapid, high in sensitivity and accuracy and good in reproducibility and stability, quantitative detection can be comprehensively and accurately conducted on the Mailuoning oral liquid, and the method has important significance on quality control of the Mailuoning oral liquid.

Description

TECHNICAL FIELD

[0001] The application relates to a quality control method of a traditional Chinese medicine preparation, in particular to a method for determining chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology. BACKGROUND

[0002] The 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, namely, 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] The pharmaceutical research of the Mailuoning oral liquid is relatively weak, and the content determination in the existing standard only detects the index component cinnamic acid in the Radix Scrophulariae, so it is difficult to accurately reflect the overall quality of the Mailuoning oral liquid. SUMMARY

[0004] The application aims at solving the problems in the prior art, and provides a method for determining chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology through a large number of experiments. The method adopts the ultra performance liquid chromatography-tandem mass spectrometry (UPLC-QqQ-MS / MS) technology, realizes the quantitative detection of 30 main chemical components, such as flavones, phenolic acids, saponins and iridoid glycosides, in the Mailuoning oral liquid while determining the cinnamic acid. The method has the characteristics of simplicity, rapidness, high sensitivity and good stability, can comprehensively and accurately quantitatively detect the Mailuoning oral liquid, and has important significance for the quality control of the Mailuoning oral liquid.

[0005] In order to achieve the above application purposes, the technical solutions provided by the application are as follows:

[0006] A method for determining chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology, comprising the following steps:

[0007] Step (1), preparation of the reference solution: a mixed reference solution of 5-hydroxymethylfurfural, harpagide, protocatechuic acid, neochlorogenic acid, loganin acid, protocatechualdehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, swertisin, seco-loganic acid, p-hydroxycinnamic acid, ferulic acid, beta-ecdysterone, jacobionoside, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isoteri-flavonoid glycoside, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, anglo-sides C, cinnamic acid, harpagide, macrostemonoside B, macrostemonoside A, asperosaponin B, ginsenoside Ro and rhizoma chuanxudan saponin IV is prepared by using methanol;

[0008] Step (2), preparation of test solution: take Maichuoning oral liquid, dilute Maichuoning oral liquid with water to 10 times the volume of Maichuoning oral liquid, filter, and take the filtrate as the test solution;

[0009] Step (3), establishment of linear regression equation: take the control solution of step (1), sequentially dilute 2 times with methanol to prepare a series of mixed control solution with different concentrations, sequentially inject into the ultra-high performance liquid chromatography-tandem mass spectrometry for detection, take the series of control solution concentrations as the abscissa and the peak area corresponding to the control solution as the ordinate, and establish the linear regression equation of each chemical component;

[0010] Step (4), detection of test solution: precisely take the test solution prepared in step (2), inject into the ultra-high performance liquid chromatography-tandem mass spectrometry for detection, substitute the peak area into the linear regression equation established in step (3), calculate the content of each chemical component in the test solution, and then calculate the content of the chemical components in Maichuoning oral liquid.

[0011] In step (1), preferably, the preparation of the control solution: respectively, 5-hydroxymethylfurfural, harpagide, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, strychnine, rhizomaroside, rhizomaroside, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteoloside, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isobiotin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, anglo C, cinnamic acid, harpagide, honeysuckle saponin B, honeysuckle saponin A, Asparagososide B, ginsenoside Ro, Chikusetsu-saponin IVA control were prepared into individual control stock solutions with methanol; respectively, 5-hydroxymethylfurfural, harpagide, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, strychnine, rhizomaroside, rhizomaroside, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteoloside, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isobiotin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, anglo C, cinnamic acid, harpagide, honeysuckle saponin B, honeysuckle saponin A, Asparagososide B, ginsenoside Ro, Chikusetsu-saponin IVA control stock solutions were mixed to obtain a mixed control solution; in the mixed control solution, the concentrations of 5-hydroxymethylfurfural, harpagide, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, strychnine, rhizomaroside, rhizomaroside, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteoloside, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isobiotin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, anglo C, cinnamic acid, harpagide, honeysuckle saponin B, honeysuckle saponin A, Asparagososide B, ginsenoside Ro, Chikusetsu-saponin IVA were respectively 10-20 μg / mL, 20-30 μg / mL, 5-10 μg / mL, 50-100 μg / mL, 30-50 μg / mL, 2-8 μg / mL, 100-120 μg / mL, 100-120 μg / mL, 15-20 μg / mL, 25-35 μg / mL, 5-10 μg / mL, 50-80 μg / mL, 110-150 μg / mL, 10-20 μg / mL, 10-20 μg / mL, 15-30 μg / mL, 25-40 μg / mL, 15-25 μg / mL, 60-70 μg / mL, 15-25 μg / mL, 50-60 μg / mL, 55-65 μg / mL, 90-100 μg / mL, 10-20 μg / mL, 10-20 μg / mL, 50-60 μg / mL, 10-20 μg / mL, 10-20 μg / mL, 10-20 μg / mL, 10-20 μg / mL.

[0012] More preferably, in the mixed control solution, the concentrations of 5-hydroxymethylfurfural, harpagide, protocatechuic acid, neochlorogenic acid, loganin acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, rhannobarside, deoxyloganin, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, juncuside, megoside, 3,4-dicaffeoylquinic acid, isomajuganin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angroside C, cinnamic acid, harpagide, macrostides B, macrostides A, notoginsenoside B, ginsenoside Ro, chikusetsusaponin IVA are 15.78 μg / mL, 28.88 μg / mL, 8.61 μg / mL, 78.60 μg / mL, 44.52 μg / mL, 5.61 μg / mL, 108.77 μg / mL, 109.63 μg / mL, 17.78 μg / mL, 29.95 μg / mL, 6.42 μg / mL, 58.82 μg / mL, 128.34 μg / mL, 14.71 μg / mL, 17.65 μg / mL, 21.39 μg / mL, 32.08 μg / mL, 19.51 μg / mL, 63.10 μg / mL, 17.25 μg / mL, 52.54 μg / mL, 58.29 μg / mL, 95.29 μg / mL, 13.64 μg / mL, 14.97 μg / mL, 55.35 μg / mL, 11.76 μg / mL, 16.17 μg / mL, 16.44 μg / mL, 14.17 μg / mL, respectively.

[0013] Further preferably, the preparation of the reference solution involves accurately weighing 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine oxycoside, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isovarenoside, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpaquinone, honeysuckle saponin B, honeysuckle saponin A, and dipsacus saponin B. Ginsenoside Ro and bamboo rhizome saponin IVA reference standards were prepared using methanol to obtain stock solutions of 5-hydroxymethylfurfural at concentrations of 1180 μg / mL, harbazoside at concentrations of 1350 μg / mL, protocatechuic acid at concentrations of 1610 μg / mL, neochlorogenic acid at concentrations of 1470 μg / mL, loganic acid at concentrations of 1110 μg / mL, protocatechuic aldehyde at concentrations of 1050 μg / mL, chlorogenic acid at concentrations of 2260 μg / mL, and cryptochlorogenic acid at concentrations of 2050 μg / mL. Vanillic acid reference stock solution 330 μg / mL, caffeic acid reference stock solution 1120 μg / mL, strychnine reference stock solution 1200 μg / mL, strychnine reference stock solution 1100 μg / mL, strychnine reference stock solution 1600 μg / mL, p-hydroxycinnamic acid reference stock solution 1100 μg / mL, ferulic acid reference stock solution 1320 μg / mL, β-ecdysterone reference stock solution 1000 μg / mL, luteolin reference stock solution 300 μg / mL, honeysuckle reference stock solution 1460 μg / mL Stock solutions of glycosides: 1180 μg / mL 3,4-dicaffeoylquinic acid, 1290 μg / mL isopropanoic acid stock solution, 1310 μg / mL 3,5-dicaffeoylquinic acid stock solution, 1090 μg / mL 4,5-dicaffeoylquinic acid stock solution, 1980 μg / mL angoloside C stock solution, 1020 μg / mL cinnamic acid stock solution, 1120 μg / mL harpagoside stock solution, 1380 μg / mL honeysuckle saponin B stock solution, 293.Stock solutions of 33 μg / mL Lonicera japonica saponin A, 1210 μg / mL Dipsacus asperoidin B, 410 μg / mL Ginsenoside Ro, and 1060 μg / mL Panax notoginseng saponin IVA were prepared. Appropriate amounts of each stock solution were precisely pipetted and mixed to obtain 5-hydroxymethylfurfural, harbazoside, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, and caffeic acid. The concentrations of the following compounds were 15.78 μg / mL and 28 μg / mL, respectively: strychnine, loganin, strychnine, oxystrychnine, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle, 3,4-dicaffeoylquinic acid, isopropanol, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpagoside, honeysuckle saponin B, honeysuckle saponin A, dipsacus saponin B, ginsenoside Ro, and bamboo ginsenoside IVA. 88μg / mL, 8.61μg / mL, 78.60μg / mL, 44.52μg / mL, 5.61μg / mL, 108.77μg / mL, 109.63μg / mL, 17.78μg / mL, 29.95μg / mL, 6.42μg / mL, 58.82μg / mL, 128.34μg / mL, 14.71μg / mL, 17.65μg / mL, 21.39μg / mL, A mixed reference solution with concentrations of 32.08 μg / mL, 19.51 μg / mL, 63.10 μg / mL, 17.25 μg / mL, 52.54 μg / mL, 58.29 μg / mL, 95.29 μg / mL, 13.64 μg / mL, 14.97 μg / mL, 55.35 μg / mL, 11.76 μg / mL, 16.17 μg / mL, 16.44 μg / mL, and 14.17 μg / mL.

[0014] The reference solution was stored at 4°C.

[0015] In step (2), preferably, the test solution is prepared by taking 1 mL of Mailuoning oral liquid into a 10 mL volumetric flask, diluting it with water to the mark, filtering it through a 0.22 μm aqueous filter membrane, and taking the filtrate as the test solution.

[0016] In steps (3) and (4), the ultra-high performance liquid chromatography (UHPLC) conditions are as follows: the chromatographic column is... UPLC HSST3C 18The chromatographic column (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size) was used. The mobile phase A was 0.1% formic acid water, and the mobile phase B was acetonitrile. The gradient elution program was as follows: 0-5 min, 5%-15% B; 5-13 min, 15%-25% B; 13-15 min, 25%-45% B; 15-16 min, 45%-100% B; 16-17.5 min, 100% B; 17.5-18 min, 100%-5% B; 18-20 min, 5% B. The flow rate was 0.3 mL / min; the column temperature was 30 °C; and the injection volume was 2 μL.

[0017] The mass spectrometry conditions were as follows: electrospray ionization (ESI) source, multiple reaction monitoring (MRM) positive and negative ion mode; cone gas flow rate 120 L / h; desolvation gas flow rate 650 L / h; desolvation temperature 650 °C; capillary voltage 3.0 kV; cone voltage and collision energy parameters are shown in the table below.

[0018]

[0019]

[0020] In step (3), the linear regression equations for each chemical component are as follows:

[0021]

[0022] The beneficial effects of this invention are:

[0023] Based on the structural characteristics of the main chemical components of Mailuoning oral liquid, such as flavonoids, phenolic acids, saponins, and iridoids, this invention screened the mass spectrometry parameters of 30 main components and used UPLC-QqQ-MS / MS technology to quantitatively determine the chemical components in Mailuoning oral liquid. Methodological evaluation showed that the method of this invention is simple, rapid, highly sensitive and accurate, reproducible, and stable, enabling comprehensive and accurate quantitative detection of Mailuoning oral liquid, which is of great significance for the quality control of Mailuoning oral liquid. Attached Figure Description

[0024] Figure 1 The total ion chromatograms of Mailuoning oral solution are shown when using different chromatographic columns.

[0025] Figure 2 The diagram shows the optimized cone voltage and collision energy of loganic acid.

[0026] Figure 3 This is the MRM ion chromatogram of the mixed standard solution.

[0027] Figure 4 MRM ion chromatogram of the test solution of Mailuoning oral liquid.

[0028] Figure 5 MRM ion chromatograms for 30 compounds. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the embodiments. It should be particularly noted that the technical solutions of the present invention have been described through preferred embodiments. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the invention without departing from its content, spirit, and scope. All similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention.

[0030] Instrument: Waters UPLC H-class series Waters TQD triple quadrupole liquid chromatography-mass spectrometry; 0.0001 g METTLER TOLEDO electronic balance.

[0031] Test drugs: 21 batches of Mailuoning oral solution, Nanjing Jinling Pharmaceutical Factory, Jinling Pharmaceutical Co., Ltd. Sample information is shown in Table 1; 5-hydroxymethylfurfural (CAS: 67-47-0), harbazoside (CAS: 6926-08-5), protocatechuic acid (CAS: 99-50-3), neochlorogenic acid (CAS: 906-33-2), loganic acid (CAS: 22255-40-9), protocatechuic aldehyde (CAS: 139-85-5), chlorogenic acid (CAS: 327-97-9), cryptochlorogenic acid (CAS: 905-9) 9-7), vanillic acid (CAS: 121-34-6), caffeic acid (CAS: 331-39-5), loganin (CAS: 18524-94-2), strychnine (CAS: 14215-86-2), oxidized loganin (CAS: 58822-47-2), p-hydroxycinnamic acid (CAS: 7400-08-0), ferulic acid (CAS: 1135-24-6), β-ecdysterone (CAS: 5289-74-7), luteolin (CAS: 5373-11-5), [unclear text - possibly related to cinnamic acid and gentianin]. 3,4-Dicaffeoylquinic acid (CAS: 14534-61-3), isopropanol (CAS: 61303-13-7), 3,5-Dicaffeoylquinic acid (CAS: 2450-53-5), 4,5-Dicaffeoylquinic acid (CAS: 32451-88-0), Angoloside C (CAS: 115909-22-3), cinnamic acid (CAS: 621-82-9), harpagoside (CAS: 19210-12-9), and gray Lonicera japonica saponin B (CAS: 136849-88-2), Lonicera japonica saponin A (CAS: 140360-29-8), Dipsacus asperoides saponin B (CAS: 33289-85-9), Ginsenoside Ro (CAS: 34367-04-9), and Panax japonicus saponin IVA (CAS: 51415-02-2) (purity ≥98%, all purchased from Chengdu Dester Biotechnology Co., Ltd.); acetonitrile (LC-MS grade), Merck; formic acid (LC-MS grade), Thermo; ultrapure water, self-made.

[0032] Table 1.21 Sample Information of Mailuoning Oral Solution

[0033]

[0034] Example 1

[0035] (1) Preparation of reference solution

[0036] Accurately weigh the following ingredients separately: 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine oxidase, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isoverostigrosin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpaquinone, honeysuckle saponin B, honeysuckle saponin A, and dicaffeoyl saponin B. Ginsenoside Ro and bamboo-root ginsenoside IVA reference standards were placed separately in volumetric flasks, dissolved in methanol, and diluted to the mark to prepare 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine glycoside, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, lonicotinic acid, 3,4-dicaffeoylquinic acid, isorhamnetin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, and angoloside C. The concentrations of cinnamic acid, harpagoside, honeysuckle saponin B, honeysuckle saponin A, dipsacus saponin B, ginsenoside Ro, and bamboo ginsenoside IVA were 1180 μg / mL, 1350 μg / mL, 1610 μg / mL, 1470 μg / mL, 1110 μg / mL, 1050 μg / mL, 2260 μg / mL, 2050 μg / mL, 1330 μg / mL, 1120 μg / mL, 1200 μg / mL, 1100 μg / mL, and 1600 μg / mL, respectively. Single reference stock solutions at concentrations of 1100 μg / mL, 1320 μg / mL, 1000 μg / mL, 300 μg / mL, 1460 μg / mL, 1180 μg / mL, 1290 μg / mL, 1310 μg / mL, 1090 μg / mL, 1980 μg / mL, 1020 μg / mL, 1120 μg / mL, 1380 μg / mL, 293.33 μg / mL, 1210 μg / mL, 410 μg / mL, and 1060 μg / mL;

[0037] Precisely aspirate the following ingredients respectively: 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine oxidase, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isovarenoside, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpaquinone, honeysuckle saponin B, honeysuckle saponin A, dipsacus saponin B, and ginsenosides. A suitable amount of Ro and bamboo rhizome saponin IVA reference stock solution were mixed to obtain 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine glycoside, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isopropanol, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpaquinone, honeysuckle saponin B, and honeysuckle saponin. The concentrations of ginsenoside A, ginsenoside B, ginsenoside Ro, and ginsenoside IVA were 15.78 μg / mL, 28.88 μg / mL, 8.61 μg / mL, 78.60 μg / mL, 44.52 μg / mL, 5.61 μg / mL, 108.77 μg / mL, 109.63 μg / mL, 17.78 μg / mL, 29.95 μg / mL, 6.42 μg / mL, 58.82 μg / mL, 128.34 μg / mL, 14.71 μg / mL, and 1, respectively. Mixed standard solutions with concentrations of 7.65 μg / mL, 21.39 μg / mL, 32.08 μg / mL, 19.51 μg / mL, 63.10 μg / mL, 17.25 μg / mL, 52.54 μg / mL, 58.29 μg / mL, 95.29 μg / mL, 13.64 μg / mL, 14.97 μg / mL, 55.35 μg / mL, 11.76 μg / mL, 16.17 μg / mL, 16.44 μg / mL, and 14.17 μg / mL were stored at 4 °C.

[0038] (2) Preparation of the test solution

[0039] Take 1 mL of each of the 21 batches of Mailuoning oral solution into a 10 mL volumetric flask, dilute with water to the mark, filter through a 0.22 μm aqueous filter membrane, and collect the filtrate to obtain the test solution.

[0040] (3) Optimization of chromatographic conditions

[0041] To achieve optimal separation of 30 chemical components, different types of chromatographic columns were investigated and compared using the test solution as the sample. Waters ACQUITY was also among the most effective. HSS T3 column (2.1×100mm, 1.8μm), Waters T3 column (2.1×100mm, 1.6μm), Waters ACQUITY BEH C 18 Chromatographic column (2.1 × 50 mm, 1.7 μm) and Waters ACQUITY BEH C 18 Four types of chromatographic columns (2.1 × 100 mm, 1.7 μm) were used. The testing conditions were as follows:

[0042] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: mobile phase A was 0.1% formic acid water, and mobile phase B was acetonitrile; the gradient elution program was as follows: 0-5 min, 5%-15% B; 5-13 min, 15%-25% B; 13-15 min, 25%-45% B; 15-16 min, 45-100% B; 16-17.5 min, 100% B; 17.5-18 min, 100%-5% B; 18-20 min, 5% B; the flow rate was 0.3 mL / min; the column temperature was 30℃; and the injection volume was 2 μL.

[0043] The mass spectrometry conditions were as follows: electrospray ionization (ESI) source, multiple reaction monitoring (MRM) positive and negative ion mode; cone gas flow rate 120 L / h; desolvation gas flow rate 650 L / h; desolvation temperature 650 °C; capillary voltage 3.0 kV; cone voltage and collision energy parameters are shown in Table 2.

[0044] Table 2. Cone voltage and collision energy parameters of 30 chemical components

[0045]

[0046]

[0047] Figure 1 The total ion chromatograms of Mailuoning oral solution using different chromatographic columns show that WatersACQUITY The HSS T3 column (2.1×100mm, 1.8μm) provided better separation of the 30 chemical components in Mailuoning oral liquid, resulting in better peak shapes. The final ultra-high performance liquid chromatography conditions were:

[0048] chromatographic column is UPLC HSS T3C 18The chromatographic column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size) was used. The mobile phase A was 0.1% formic acid water, and the mobile phase B was acetonitrile. The gradient elution program was as follows: 0-5 min, 5%-15% B; 5-13 min, 15%-25% B; 13-15 min, 25%-45% B; 15-16 min, 45%-100% B; 16-17.5 min, 100% B; 17.5-18 min, 100%-5% B; 18-20 min, 5% B. The flow rate was 0.3 mL / min; the column temperature was 30 °C; and the injection volume was 2 μL.

[0049] (4) Mass spectrometry parameter optimization

[0050] To obtain the optimal response of the chemical components, the optimal cone voltage and collision energy for 30 chemical components were investigated, comparing different cone voltages (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 V) and different collision energies (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 V). Loganiic acid was used as an example (see...). Figure 2 Loganic acid showed the highest peak response at a cone voltage of 40V and a collision energy of 20V. Similarly, the optimal cone voltage and collision energy for the other components are shown in Table 3.

[0051] The optimal mass spectrometry conditions were: electrospray ionization (ESI) source, multiple reaction monitoring (MRM) positive and negative ion mode; cone gas flow rate 120 L / h; desolvation gas flow rate 650 L / h; desolvation temperature 650 °C; capillary voltage 3.0 kV; cone voltage and collision energy parameters are shown in Table 2.

[0052] Table 3. MRM mass spectrometry parameters of 30 compounds

[0053]

[0054]

[0055] (5) Establish the linear regression equation

[0056] Take the mixed reference solution prepared in “(1) Preparation of reference solution”, dilute it with methanol by 2 times in sequence, dilute it for a total of 15 points, and prepare a series of mixed reference solutions of different concentrations. Inject the solutions into the liquid chromatography-mass spectrometry instrument in sequence and detect them according to the chromatographic and mass spectrometry conditions in (3) and (4).

[0057] Using the concentrations of a series of reference standards as the x-axis and the corresponding peak areas as the y-axis, regression analysis was performed on each chemical component to obtain the linear regression equation and correlation coefficient (r). 2 The linear range is shown in Table 4.

[0058] Table 4. Linear regression equations and correlation coefficients (r) for 30 compounds. 2 ), linear range, limit of detection, and limit of quantitation

[0059]

[0060]

[0061] (6) Methodological investigation

[0062] Precision: The mixed reference solution prepared in “(1) Preparation of reference solution” was injected 6 times consecutively on the first day, the second day and the third day according to the optimal chromatographic conditions determined in (3) and the optimal quality chromatographic conditions determined in (4). The peak areas of 30 chemical components were measured and their relative standard deviations (RSD%) were calculated. The results are shown in Table 5.

[0063] Repeatability: Take the oral liquid of Mailuoning and prepare 6 test solutions in parallel according to "(2) Preparation of test solution". According to the optimal chromatographic conditions determined in (3) and the optimal quality chromatographic conditions determined in (4), the samples were injected and the peak areas of 30 chemical components were determined and their relative standard deviations (RSD%) were calculated. The results are shown in Table 5.

[0064] Stability: Take the oral liquid of Mailuoning and prepare the test solution according to "(2) Preparation of test solution". According to the optimal chromatographic conditions determined in (3) and the optimal quality chromatographic conditions determined in (4), the peak areas of 30 chemical components were determined at 0, 3, 6, 9, 12, 24 and 33 h, respectively, and their relative standard deviations (RSD%) were calculated. The results are shown in Table 5.

[0065] Recovery rate: Take 9 portions of Mailuoning oral liquid in parallel. Add the corresponding reference standards according to 50%, 100% and 150% of the component content in the sample. According to the optimal chromatographic conditions determined in (3) and the optimal quality chromatographic conditions determined in (4), inject the sample to determine the peak area of ​​30 chemical components and calculate their recovery rate and relative standard deviation (RSD%). The results are shown in Table 5.

[0066] Table 5. Precision, repeatability, stability, and recovery rate

[0067]

[0068]

[0069] (7) Detection of the test solution

[0070] The 21 batches of test solutions prepared in “(2) Preparation of test solution” were precisely sampled and injected into the liquid chromatography-mass spectrometry instrument for detection according to the optimal chromatographic conditions determined in (3) and the optimal quality spectral conditions determined in (4). The contents of 30 chemical components in each test solution were calculated by substituting into the linear regression equation established in (5), and then the contents of chemical components in Mailuoning oral liquid were calculated. The results are shown in Table 6 and Table 7.

[0071] (8) Results Analysis

[0072] The content determination results of the above 21 batches of Mailuoning oral solution show that there are differences between different batches. For example, the total content of 30 analytes in Mailuoning oral solution varies slightly between 3.70 and 4.22 mg / mL. The contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid vary between 0.52 and 0.61 mg / mL, 0.50 and 0.59 mg / mL, and 0.41 and 0.49 mg / mL, respectively, which are higher than those of the other 27 compounds.

[0073] The above results show that the method established in this invention can simultaneously determine the content of 30 components in Mailuoning oral liquid. Furthermore, this method is simple, rapid, and has the characteristics of high sensitivity and accuracy, good reproducibility and stability. It can comprehensively and accurately perform quantitative detection of Mailuoning oral liquid, which is of great significance for the quality control of Mailuoning oral liquid.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0075]

[0076]

[0077]

Claims

1. A method for determining the chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology, characterized in that: Includes the following steps: Step (1) Preparation of reference solution: A mixed reference solution of 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine oxidase, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isopropanol, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpaquinone, honeysuckle saponin B, honeysuckle saponin A, dipsacus saponin B, ginsenoside Ro, and bamboo ginsenoside IVA was prepared using methanol. Step (2), preparation of test solution: Take an appropriate amount of Mailuoning oral liquid, dilute Mailuoning oral liquid with water to 10 times the volume of Mailuoning oral liquid, filter, and take the filtrate as test solution; Step (3) Establishment of linear regression equation: Take the reference solution from step (1), dilute it twice with methanol to prepare a series of mixed reference solutions of different concentrations, and inject them into an ultra-high performance liquid chromatography-tandem mass spectrometry instrument for detection. Use the concentration of the series of references as the abscissa and the peak area corresponding to the references as the ordinate to establish the linear regression equation for each chemical component. Step (4) Detection of the test solution: Accurately pipette the test solution prepared in step (2) and inject it into an ultra-high performance liquid chromatography-tandem mass spectrometry instrument for detection. Substitute the peak area into the linear regression equation established in step (3) to calculate the content of each chemical component in the test solution, and then convert it to obtain the content of chemical components in the Mailuoning oral liquid.

2. The method for determining the chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology according to claim 1, characterized in that: In step (1), the preparation of the reference solution is as follows: 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine oxytocin, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isopropanol, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpaquinone, honeysuckle saponin B, honeysuckle saponin A, dipsacus saponin B, ginsenoside Ro, and bamboo ginsenoside IVA reference standards are accurately weighed and prepared into individual reference stock solutions using methanol; 5-hydroxymethylfurfural is accurately pipetted into the following solutions. Aldehyde, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine oxidase, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isobarbituronide, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpaquinone, honeysuckle saponin B, honeysuckle saponin A, dipsacus saponin B, ginsenoside Ro, and bamboo ginsenoside IVA reference standard stock solutions were mixed to obtain a mixed reference solution; in the mixed reference solution, 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, and chlorogenic acid were present. The concentrations of the following compounds were observed: chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine oxidase, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isopropanol, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpagoside, lysimachia christinae B, lysimachia christinae A, dapoxetine B, ginsenoside Ro, and ginsenoside IVA were 10–20 μg / mL, 20–30 μg / mL, 5–10 μg / mL, 50–100 μg / mL, 30–50 μg / mL, 2–8 μg / mL, 100–120 μg / mL, and 100–120 μg / mL, respectively. g / mL, 15~20μg / mL, 25~35μg / mL, 5~10μg / mL, 50~80μg / mL, 110~150μg / mL, 10~20μg / mL, 10~20μg / mL, 15~30μg / mL, 25~40μg / mL, 15~25μg / mL, 60~70μg / mL, 15~25μg / mL, 50~60μg / mL, 55~65μg / mL, 90~100μg / mL, 10~20μg / mL, 10~20μg / mL, 50~60μg / mL, 10~20μg / mL, 10~20μg / mL, 10~20μg / mL, 10~20μg / mL.

3. The method for determining the chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology according to claim 2, characterized in that: The mixed reference solution contains 5-hydroxymethylfurfural, harpaquinone, protocatechuic acid, neochlorogenic acid, loganic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, vanillic acid, caffeic acid, loganin, strychnine, strychnine glycoside, p-hydroxycinnamic acid, ferulic acid, β-ecdysterone, luteolin, honeysuckle glycoside, 3,4-dicaffeoylquinic acid, isorhamnetin, and 3,5-dicaffeoylquinic acid. The concentrations of 4,5-dicaffeoylquinic acid, angoloside C, cinnamic acid, harpagoside, honeysuckle saponin B, honeysuckle saponin A, dipsacus saponin B, ginsenoside Ro, and bamboo ginsenoside IVA were 15.78 μg / mL, 28.88 μg / mL, 8.61 μg / mL, 78.60 μg / mL, 44.52 μg / mL, and 5.61 μg / mL, respectively. mL, 108.77μg / mL, 109.63μg / mL, 17.78μg / mL, 29.95μg / mL, 6.42μg / mL, 58.82μg / mL, 128.34μg / mL, 14.71μg / mL, 17.65μg / mL, 21.39μg / mL, 32.08μg / mL, 19.51μg / mL, 63.10μg / mL, 17.25μg / mL, 52.54μg / mL, 58.29μg / mL, 95.29μg / mL, 13.64μg / m L, 14.97μg / mL, 55.35μg / mL, 11.76μg / mL, 16.17μg / mL, 16.44μg / mL, 14.17μg / mL.

4. The method for determining the chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology according to claim 1, characterized in that: In step (2), the preparation of the test solution is as follows: take 1 mL of Mailuoning oral liquid into a 10 mL volumetric flask, dilute with water to the mark, filter through a 0.22 μm aqueous filter membrane, and take the filtrate as the test solution.

5. The method for determining the chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology according to claim 1, characterized in that: The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: chromatographic column: UPLC HSS T3 C 18 The chromatographic column was 100 mm long, 2.1 mm in inner diameter, and had a particle size of 1.8 μm. The mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B). The gradient elution program was as follows: 0–5 min, 5%–15% B; 5–13 min, 15%–25% B; 13–15 min, 25%–45% B; 15–16 min, 45%–100% B; 16–17.5 min, 100% B; 17.5–18 min, 100%–5% B; 18–20 min, 5% B. The flow rate was 0.3 mL / min; the column temperature was 30 °C; and the injection volume was 2 μL.

6. The method for determining the chemical components in Mailuoning oral liquid based on UPLC-QqQ-MS / MS technology according to claim 1 or 5, characterized in that: Mass spectrometry conditions were as follows: electrospray ionization source, multiple reaction monitoring (MRM) positive / negative ion mode; cone gas flow rate 120 L / h; desolvation gas flow rate 650 L / h; desolvation temperature 650 °C; capillary voltage 3.0 kV; cone voltage and collision energy parameters are as follows:

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