Chemical component systematic characterization method, quality marker, detection method and application of ginseng root strengthening oral liquid

Abstract

The invention relates to the technical field of Chinese patent medicine analysis, in particular to a chemical component systematic characterization method, a quality marker, a detection method and application of ginseng root strengthening oral liquid. According to the chemical component systematic characterization method provided by the invention, the chemical components in the ginseng root-consolidating oral liquid are detected by combining offline two-dimensional liquid chromatography and quadrupole rod high-resolution mass spectrometry, and the chemical component systematic characterization method not only can be used for systematic characterization of each component, but also can be used for further screening mass markers, so that the quality control of the ginseng root-consolidating oral liquid is favorably realized. The quality marker provided by the invention covers eight traditional Chinese medicines and eight structural types of compounds of the ginseng root-consolidating oral liquid, and can be used for overall quality control of the ginseng root-consolidating oral liquid. The detection method provided by the invention can be used for simply, quickly and quantitatively detecting the quality markers of the ginseng root strengthening oral liquid, and has very strong operability and practicability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine analysis, and in particular to a chemical component systematic characterization method of Radix Ginseng Oral Liquid, a quality marker and a detection method and application thereof. BACKGROUND

[0002] Traditional Chinese medicine prescriptions are the most common form in clinical practice. Due to the combination of multiple herbal ingredients, its complexity usually leads to the diversity of chemical components, including various structures, numerous isomers and significant differences in content and polarity. Clarifying the composition of compounds and distinguishing the true and false are the core of traditional Chinese medicine quality control, which is crucial for ensuring the therapeutic effect.

[0003] Radix Ginseng Oral Liquid (RSGB) is a traditional Chinese medicine widely used in clinical practice, which has the effect of immune regulation and can effectively improve the clinical indicators of cancer patients during postoperative period and adjuvant chemotherapy. It is a complex traditional Chinese medicine composed of ten Chinese herbal medicines, namely Rehmannia glutinosa, Radix Rehmanniae Praeparatae, Radix Ginseng, Dioscorea Opposita, Fructus Corni, Ophiopogon, Radix Asparagi, Poria, Alisma and Cortex Moutan. Its composition is very complex, mainly including ginsenosides, phenols, terpenes, sugars and amino acids. The existing literature on RSGB mainly focuses on its mechanism of action, and the composition research usually only targets the content determination or thin layer chromatography identification of single herbal ingredients, which cannot fully reflect the chemical components and overall quality of RSGB. Therefore, it is necessary to establish a more scientific and comprehensive chemical component characterization method and quality control method for RSGB. SUMMARY

[0004] In view of the above technical problems, the present application provides a chemical component systematic characterization method of Radix Ginseng Oral Liquid, a quality marker and a detection method and application thereof. The chemical component systematic characterization method can detect more than 200 chemical components in Radix Ginseng Oral Liquid, which can not only be used for systematic characterization of each component, but also be used for further screening of quality markers, thereby helping to realize the quality control of Radix Ginseng Oral Liquid. The quality marker provided by the present application covers 8 kinds of Chinese medicines and 8 kinds of structural compounds of Radix Ginseng Oral Liquid, which provides a reference on strategy and method for the overall quality control of Radix Ginseng Oral Liquid. The detection method of the above quality marker provided by the present application can quantitatively detect the above quality marker with large content difference in Radix Ginseng Oral Liquid simply and quickly, which has strong operability and practicability.

[0005] In order to achieve the above application purposes, the embodiments of the present application adopt the following technical solutions:

[0006] The first aspect of the present application provides a chemical component systematic characterization method of ginseng oral liquid, which uses offline two-dimensional liquid chromatography (2D-LC) combined with quadrupole high-resolution mass spectrometry to detect chemical components in the ginseng oral liquid; the first dimension chromatography in the offline two-dimensional liquid chromatography is hydrophilic interaction chromatography, and the second dimension chromatography is reversed-phase chromatography.

[0007] In the offline two-dimensional liquid chromatography of the present application, the hydrophilic mode of the first dimension chromatography has certain separation capacity and convenient post-treatment, and can be used for initial separation of complex chemical components in the ginseng oral liquid, and effectively obtain components suitable for further analysis; the second dimension chromatography is a reversed-phase mode with high separation efficiency and stability, which can maximize the capture of compounds. The present application combines offline two-dimensional liquid chromatography with quadrupole high-resolution mass spectrometry for qualitative and semi-quantitative analysis, which not only increases the separation degree of the components to be detected in the ginseng oral liquid, but also improves the sensitivity of trace compounds through offline enrichment method, so that the number of detected components is significantly improved compared with the prior art, and the types and relative contents of small molecule components in the ginseng oral liquid can be systematically and comprehensively analyzed.

[0008] Preferably, the chromatographic conditions of the first dimension chromatography are as follows:

[0009] Chromatographic column: octadecylsilane-bonded silica gel chromatographic column;

[0010] The mobile phase A is 9-11 mM ammonium acetate aqueous solution, and the mobile phase B is acetonitrile, and the linear gradient elution program is as follows:

[0011]

[0012] Flow rate: 1.0 mL / min;

[0013] From 0 to 27 min, the eluent collected every 3 min is a fraction, and the eluent collected from 27 to 32 min is a fraction; after drying each fraction, dissolve in 50% v / v methanol, centrifuge, and take the supernatant for second dimension chromatography analysis;

[0014] The chromatographic conditions of the second dimension chromatography are as follows:

[0015] Chromatographic column: reversed-phase chromatographic column;

[0016] The mobile phase A is 0.1% v / v formic acid aqueous solution, and the mobile phase B is acetonitrile;

[0017] The gradient elution program is as follows:

[0018]

[0019] Flow rate 0.3 mL / min.

[0020] Preferably, in the chromatographic conditions of the first dimension chromatography, the column is Acchrom XAmide, 4.6 x 150 mm, 5 pm.

[0021] Preferably, in the chromatographic conditions of the first dimension chromatography, the mobile phase A is 10 mM ammonium acetate in water.

[0022] Preferably, in the chromatographic conditions of the first dimension chromatography, the column temperature is 25-45 °C. Further preferably, 35 °C. The separation of chromatographic peaks is most obvious at 35 °C, especially for the compounds with lower response detected within 13-16 min.

[0023] Different reversed-phase packing materials have different separation selectivity. Preferably, in the chromatographic conditions of the second dimension chromatography, the column is CSH Phenyl-Hexyl, CSH C18 and BEH Shield RP18, 2.1 x 100 mm, 1.7 pm. The above three reversed-phase columns have greater peak capacity in terms of the number of chromatographic peaks, resolution and the number of full scan primary fragments.

[0024] Further preferably, the column in the second dimension chromatography is BEH Shield RP18. BEH Shield RP18 is a reversed-phase column that changes polarity by bonding hydrophilic carbamate functional groups on C18, which can tolerate 100% water and maintain stability in a wide pH range of pH 2 to 11. Within the first 3 min of the chromatogram, there are some poorly retained compounds with high polarity. BEH Shield RP18 can provide better retention and separation for these compounds.

[0025] Preferably, in the chromatographic conditions of the second dimension chromatography, the column temperature is 25-45 °C. Further preferably, 45 °C.

[0026] Preferably, the quadrupole high-resolution mass spectrometer is Exploris 120 Q-Orbitrap mass spectrometer equipped with HESI source, and data is collected in positive and negative ion switching mode. Ginsenosides and iridoids have enhanced response in negative ion mode, while phenols and amino acids have higher response area in positive ion mode, so full scan mode with HESI+ and HESI- is selected.

[0027] Preferably, the parameters of the mass spectrometer are: spray voltage, -3.0 kV / +2.5 kV; sheath gas pressure, 35 arb; auxiliary gas pressure, 10 arb; sweep gas pressure, 0 arb; capillary temperature, 350℃; auxiliary gas temperature, 400℃; resolution of the first and second stages is 60000 and 15000 respectively; scan range m / z 100-1500, AGC is set to 1x10 6 and 1x10 5 ; MS 1 The top 4 ions of medium intensity are automatically selected to trigger high-energy collision-induced dissociation (HCD) MS 2 fragmentation; the normalized collision energy (NCE) is set to 10-20 / 20-40 / 30-60 V, and the isolation width is 4.0 Da; the dynamic exclusion time is set to 6S.

[0028] Preferably, the normalized collision energy is set to 10 / 30 / 50 V

[0029] The second aspect of the present application provides an application of the above-mentioned chemical component system characterization method in screening quality markers (Q-Markers) of Renshen Guben Oral Liquid: through network pharmacology, active ingredients related to immune function are screened from the compounds identified by the above-mentioned chemical component system characterization method, and the quality markers are further screened by combining the fingerprint of the traditional Chinese medicine components of Renshen Guben Oral Liquid.

[0030] The third aspect of the present application provides a group of quality markers of Renshen Guben Oral Liquid, which includes: catalpol, acteoside, Reginsenoside Rg1, Reginsenoside Rb1, Reginsenoside Re, loganin, paeonol, paeoniflorin, gallic acid, Alisol B, asparagine and adenosine.

[0031] The above-mentioned quality markers are firstly screened from the compounds identified by the above-mentioned chemical component system characterization method through network pharmacology, and then further screened by combining the fingerprint of the traditional Chinese medicine components of Renshen Guben Oral Liquid and the safety of the compounds.

[0032] For the monarch drug Rehmannia glutinosa (RR) and Radix Rehmanniae Preparata (RRP), catalpol and acteoside are relatively abundant compounds, and according to the Chinese Pharmacopoeia (2020 edition), both are included in the quality control indicators.

[0033] In the selection of Q-Marker, the principle of monarch, minister, assistant and messenger should be considered, and the minister herbs (such as ginseng (GR), cornus fruit (CF) and cortex moutan (MC)) and other five kinds of assistant and messenger herbs are also important. It is also necessary to control the quality of ginseng as a precious medicinal material. The ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re are specified in the Chinese Pharmacopoeia as quality control indicators. According to the common peaks in the RSGB and the fingerprint of the constituent herbs and the quality control components in the Chinese Pharmacopoeia, the quality of cornus fruit is controlled by loganin and gallic acid, and the quality of cortex moutan is controlled by gallic acid, paeonol and paeoniflorin. Gallic acid has a high content in the oral solution and shows significant anti-tumor and cardiovascular protection pharmacological effects, so it is used as an investigation indicator for cornus fruit and cortex moutan. Alisol B is a potential active ingredient screened by network pharmacology, and has strong specificity, only from alisma (ALR) medicinal materials, and can also be reflected in the fingerprint, so the quality of alisma is controlled by alisol B. The above chemical components are included in the quality control indicators.

[0034] Asparagus (ASR) and dioscorea (DR) do not have specified markers in the Chinese Pharmacopoeia, and based on the fingerprint, asparagine and adenosine are selected to reflect the quality change process, respectively.

[0035] Due to the low polarity, the components in ophiopogon (OR) and poria (PR) have low extraction efficiency under the current process conditions, and it is difficult to find their individual characteristic components, so the Q-Marker is not selected from them.

[0036] The above quality markers ensure the traceability of the whole production process of ginseng solid root oral solution and the quality transmission from raw materials to final products.

[0037] The fourth aspect of the present application provides a detection method of the above quality markers: using the method of liquid chromatography-mass spectrometry to analyze the quality markers;

[0038] The liquid chromatography condition in the liquid chromatography-mass spectrometry is:

[0039] The chromatographic column is an octadecylsilane bonded silica gel chromatographic column;

[0040] The mobile phase A is 0.1% v / v formic acid aqueous solution, the mobile phase B is acetonitrile, and the linear gradient elution program is:

[0041]

[0042]

[0043] The flow rate is 0.3 mL / min;

[0044] The mass spectrometry in the liquid chromatography-mass spectrometry adopts an adjustable multiple reaction monitoring (MRM) mode.

[0045] The prior art for quality control of ginseng solid base at present is mainly the detection of single medicinal material or single component in preparation, such as, four thin layer chromatography methods are used to identify four Chinese medicines of ginseng, cornus, cortex moutan and alisma in preparation, three HPLC methods are used to respectively determine the contents of ginsenoside Rg1 and Re of ginseng, paeonol of cortex moutan and loganin and loganin of cornus in preparation, which cannot comprehensively control the quality of ginseng solid base oral liquid, and the sample pretreatment needs to be extracted, dried and the like.

[0046] The contents of the above-mentioned 12 Q-Markers in ginseng solid base oral liquid are greatly different, the concentrations of gallic acid and paeonol exceed the detection limit of the instrument, and alismol B is close to the lower limit of quantification (LOQ), the content difference of the compound with the highest and lowest contents is as high as 50,000 times, and the quantitative results of each compound usually need to be respectively detected. The above-mentioned detection method provided by the application can realize the simultaneous determination of the above-mentioned 12 Q-Markers covering 8 Chinese medicines in 10 min, greatly improving the detection efficiency and the quality control coverage of medicinal materials, and the sample pretreatment only needs simple dilution. Compared with the prior art, the detection method is simple and feasible, and is more convenient for the overall quality control of ginseng solid base oral liquid.

[0047] Preferably, the chromatographic column is BEH Shield RP18 with a specification of 2.1x100mm, 1.7μm.

[0048] Preferably, the column temperature is 35-45℃. Further preferably, 40℃.

[0049] Preferably, the mass spectrometer in the liquid chromatograph-mass spectrometer is Xevo TQ-S mass spectrometer.

[0050] Preferably, the mass spectrometer sets the parent ion, daughter ion, orifice voltage, collision energy and acquisition mode for each mass marker as follows:

[0051]

[0052]

[0053] Preferably, the parameters of the mass spectrometer further include: capillary voltage, +3.0kV / -2.8kV; desolvation gas (N2) temperature and flow rate are 500℃ and 1000L / h respectively.

[0054] The application adopts adjustable MRM mode to adapt to compounds with different content ranges, reduces the response of high-abundance compounds by using suboptimal parameters (ion pair with non-maximum peak area, orifice voltage, collision energy), and enhances the response of trace compounds by using optimal parameters (ion pair with maximum peak area, orifice voltage, collision energy). Finally, by using weak collision energy and ion pair parameters, the response of gallic acid and paeonol is weakened, and the upper limit of quantification is expanded, thereby providing an effective solution for comprehensive and systematic evaluation of the quality of Renshen Guben oral liquid.

[0055] The application has the beneficial effects that: the application firstly uses off-line two-dimensional liquid chromatography combined with quadrupole high-resolution mass spectrometry to comprehensively qualitatively and quantitatively evaluate RSGB. Through specific chromatographic conditions and mass spectrometric conditions, the resolution of the to-be-measured components in Renshen Guben oral liquid is increased, and the sensitivity of trace compounds is improved through the off-line enrichment method, so that the types and relative contents of small molecule components in Renshen Guben oral liquid are more systematically and comprehensively analyzed, and finally 275 compounds are separated and identified from RSGB, and the number of detected components is obviously improved compared with the prior art. Through network pharmacology, potential active ingredients related to immunity are explored, combined with the fingerprint of ten component herbs and important components, the application selects a total of 12 compounds from 8 different traditional Chinese medicines and 8 different categories of compounds as quality markers, which provides a reference on strategy and method for the overall quality control of RSGB. The contents of the 12 compounds in RSGB are quite different, and the application realizes the simultaneous quantification of the 12 compounds by using adjustable multiple reaction monitoring method, and the sample pretreatment is simple, which provides a simple and feasible method for the overall quality control of RSGB. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 Structural formulas of 52 control samples used in the examples;

[0057] Figure 2 Orthogonal evaluation results of the off-line two-dimensional liquid chromatography system in Example 1;

[0058] Figure 3 Chromatograms of 10 fractions of RSGB prepared based on the first dimension in Example 1;

[0059] Figure 4 Mass spectrometric fragmentation rules of loganin, ginsenoside Rg3 and paeoniflorin in Example 1;

[0060] Figure 5 Structural classification and number of compounds in RSGB in Example 1;

[0061] Figure 6 Compound-target-pathway analysis network in Example 4;

[0062] Figure 7 UPLC-MS fingerprint combination of RSGB and 8 ingredients in Example 5 and Q-Marker;

[0063] Figure 8 Extracted ion chromatograms of 12 Q-Markers in blank solvent (A), mixed reference solution (B) and RSGB oral solution (C) in Example 6;

[0064] Figure 9 Content determination results of 10 batches of RSGB in Example 6;

[0065] Figure 10 Chromatograms at 270 (left) and 203 nm (right) obtained by the first dimension chromatography at different column temperatures in Test Example 1;

[0066] Figure 11 Base peak chromatograms obtained by the second dimension chromatography at different column temperatures in Test Example 2;

[0067] Figure 12 Abundance of characteristic fragment ions produced by different compounds at different collision energies in the mass spectrometry in Test Example 3;

[0068] Figure 13 Chromatograms at 270 (left) and 203 nm (right) obtained by the first dimension chromatography using different aqueous phases in Test Example 4;

[0069] Figure 14 Scatter plot of target analytes (A) and chromatograms at 270 (B1) and 203 nm (B2) obtained by the first dimension chromatography using different chromatographic columns in Test Example 5;

[0070] Figure 15 SIEVE extracted primary scatter plot (A) and base peak chromatogram (B) obtained by the second dimension chromatography using different aqueous phases in Test Example 6;

[0071] Figure 16 Extracted chromatogram (A) and SIEVE extracted primary scatter plot (B) obtained by the second dimension chromatography using different chromatographic columns (C) in Test Example 7;

[0072] Figure 17 Peak area of index components at different spray voltages in the mass spectrometry in Test Example 8;

[0073] Figure 18 Peak area of index components at different capillary temperatures in the mass spectrometry in Test Example 9;

[0074] Figure 19 Peak area of index components at different auxiliary gas temperatures in the mass spectrometry in Test Example 10. Detailed Implementation

[0075] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0076] Renshen Guben Oral Liquid (RSGB) is made from ten kinds of traditional Chinese medicine, and its composition is very complex. Systematic characterization and comprehensive quantification of chemical components are prerequisites for elucidating the pharmacological basis and quality control of traditional Chinese medicine. However, current research on the components of RSGB usually only focuses on the content determination or thin-layer chromatography identification of single herbal components, which cannot fully reflect the overall quality of RSGB.

[0077] To address this issue, this invention provides a method for systematically characterizing the chemical components of ginseng oral liquid using RSGB. This method uses offline two-dimensional liquid chromatography (2D-LC) coupled with quadrupole high-resolution mass spectrometry to detect the chemical components in ginseng tonic oral liquid. First, the complex chemical components in ginseng tonic oral liquid are effectively separated and captured under specific chromatographic conditions. Then, quadrupole high-resolution mass spectrometry is used for qualitative and semi-quantitative analysis, thereby achieving systematic characterization of small molecule components in ginseng tonic oral liquid. The number of components detected is significantly improved compared with existing technologies.

[0078] The embodiments of the present invention also provide the application of the above-mentioned chemical component system characterization method in screening quality markers (Q-Markers) for ginseng tonic oral liquid, and provide a set of Q-Markers that can be used to monitor the RSGB production process and product quality.

[0079] The present invention also provides a detection method for analyzing the above-mentioned Q-Marker using liquid chromatography-mass spectrometry (LC-MS) technology.

[0080] The technical solution of the present invention will be further described below through specific embodiments.

[0081] The reagents and chemicals used in the following examples:

[0082] 52 reference standards (structure as follows) Figure 1 As shown), it includes 10 triterpenes, 7 phenols, 8 iridoids, 5 organic acids, 5 amino acids, 3 flavonoids, 3 nucleosides, 2 phenylethanol, 4 sugars, 2 steroids, 1 terpene and 2 other compounds, which were purchased from Shanghai Biaode Biotechnology Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd. or Chengdu Dester Biotechnology Co., Ltd., respectively.

[0083] Acetonitrile, methanol (Fisher, Fair lawn, NJ, USA), ammonium formate (AF, Aladdin, Shanghai) were HPLC grade, ammonium acetate (AA, Fisher), acetic acid (AC, ACS reagent, Sigma Aldrich, St. Louis, MO, USA) and formic acid (FA, ACS) were LC-MS grade;

[0084] Milli-Q A10 water purification system (Millipore, Bedford, MA, USA) was used to prepare ultrapure water;

[0085] Ten batches of Renshen Guben oral liquid (No. S1-S10, batch number: 329210452, 329210482, 329230042, 329230102, 329230112, 329230122, 329230132, 329230142, 329230152, 329230212) were provided by Lunan Houpu Pharmaceutical Co., Ltd. and identified by Professor Zhang Lijuan of Tianjin University of Chinese Medicine.

[0086] Example 1

[0087] This example provides a method for the systematic characterization of the chemical components of Renshen Guben oral liquid.

[0088] 1. Preparation of sample solution

[0089] Ten batches of RSGB samples were diluted 10 times with 50% v / v methanol and then centrifuged at 20817g at 4°C for 10 min. The supernatant after centrifugation was stored in a 4°C refrigerator for use.

[0090] Ten traditional Chinese medicines were respectively crushed and passed through a No. 4 sieve. About 10 mg of powder was weighed for each and added to 1 mL of 50% methanol-water (v:v) for ultrasonic extraction for 1 h. Centrifugation was performed at 20817g at 4°C for 10 min, and the supernatant after centrifugation was stored in a 4°C refrigerator for use.

[0091] 2. Off-line two-dimensional liquid chromatography system conditions

[0092] 2.1 First-dimensional hydrophilic interaction chromatography conditions:

[0093] RSGB was initially separated using a Waters e2695 HPLC system and an Acchrom XAmide column (4.6 × 150 mm, 5 μm). The mobile phase was 10 mM ammonium acetate aqueous solution (A)-acetonitrile (B) at a flow rate of 1.0 mL / min; the column temperature was 35 °C; the detector was a Waters 2489 UV detector at wavelengths (203 and 270 nm); and the injection volume was 20 μL. The gradient elution program was as follows:

[0094]

[0095] The eluent was divided into 10 time intervals: liquid was collected every 3 minutes from 0 to 27 min as one fraction, and collected from 27 to 32 min as the 10th fraction. The combined liquid of each fraction was dried under nitrogen at room temperature. The residues were redissolved in 200 μL of 50% v / v methanol, vortexed for 3 min, centrifuged at 20817 g and 4 °C for 10 min, and the supernatant was used for the second dimension analysis.

[0096] 2.2 Second-dimensional reversed-phase chromatographic conditions:

[0097] The second dimension was performed on a Thermo Ultimate 3000U HPLC system. The column was a BEH Shield RP18 (2.1 × 100 mm, 1.7 μm; Waters), column temperature 45 °C; mobile phase was 0.1% v / v formic acid aqueous solution (A) - acetonitrile (B), flow rate 0.3 mL / min; injection volume 2 μL; gradient elution program as follows:

[0098]

[0099] 3. Mass spectrometry conditions:

[0100] A Thermo Exploris 120Q-Orbitrap mass spectrometer equipped with a HESI source was used. Data was acquired in positive and negative ion switching mode. The parameters were set as follows: spray voltage, -3.0 kV / +2.5 kV; sheath gas pressure, 35 arb; auxiliary gas pressure, 10 arb; purge gas pressure, 0 arb; capillary temperature, 350 °C; auxiliary gas temperature, 400 °C; primary and secondary resolutions were 60,000 and 15,000, respectively; scan range m / z 100-1500; AGC was set to 1×10⁻⁶. 6 and 1×10 5 MS 1 MS with the four strongest ions automatically selected to trigger high-energy collision-induced dissociation (HCD) 2Fragmentation. Normalized collision energy (NCE) was set to 10 / 30 / 50 V, isolation width was 4.0 Da. Dynamic exclusion time was set to 6 S.

[0101] 4. Methodology Investigation

[0102] The repeatability and precision of the first dimension and the second dimension chromatography system were investigated, and the results are shown in Table 1 and Table 2. All the results meet the requirements of the methodology.

[0103] Table 1 Methodology Investigation Results of the First Dimension Chromatography

[0104]

[0105] Table 2 Methodology Investigation Results of the Second Dimension Chromatography

[0106]

[0107] 5. Evaluation of the Two-Dimensional Chromatography System

[0108] The performance of the two-dimensional chromatography system was evaluated from the aspects of orthogonality and peak capacity.

[0109] The orthogonality of the system was evaluated by the star plot method. The retention times of 50 target components in both dimensions were determined, and the compound distribution map was constructed by the normalized relative retention time (see Figure 2 ). These components were mainly distributed in four different lines, represented by Z-, Z+, Z1 and Z2, and the corresponding Z values were 1.00, 0.71, 0.85 and 0.89, respectively. The size of the Z value is highly related to the orthogonality between the two chromatographic separation mechanisms; a higher Z value indicates better orthogonality. The overall orthogonality value (A0) of the 2D-LC system was 0.73, indicating good separation orthogonality.

[0110] The peak capacity of the system is the product of the peak capacity of its one dimension (1D) and two dimensions (2D), and the system has the maximum peak capacity under the condition of complete orthogonality. The peak widths of 9 chromatographic peaks in the front, middle and rear sections of the effective elution time in both dimensions were recorded, the average chromatographic peak width was calculated, and the peak capacity of each dimension was calculated by dividing the total elution time by the average peak width. For one-dimensional liquid chromatography, the average peak width was 0.66 min, the total elution time was 32 min, and the peak capacity was 48. While for the second dimension chromatography, the average peak width was 0.32 min, the total elution time was 40 min, and the peak capacity was 125. Therefore, the peak capacity of the constructed two-dimensional liquid system was 6000, which was about 50 times higher than that of the traditional one-dimensional liquid chromatography.

[0111] 6. Multi-component characterization of the ginseng oral liquid

[0112] The data acquisition and analysis results of the 10 fraction samples of the ginseng oral liquid are shown in Table 1. Figure 3

[0113] The RSGB contains compounds of various structures, such as iridoid, triterpene and sesquiterpene. Taking the three as examples, the cleavage rules are shown in Table 2. Figure 4

[0114] MS 2 The data were used for structure identification and characterization. By comparison with reference substances, literature retrieval, and matching with the mzCloud library and the HMDB public database with an error within 5ppm, a total of 275 compounds were identified, involving 13 types of compounds (as shown in Table 3). 77 and 198 compounds were identified in positive and negative ion modes, respectively. Figure 5

[0115] Example 2

[0116] The present embodiment provides a method for systematically characterizing the chemical components of the ginseng oral liquid.

[0117] 1. Preparation of sample solution: same as in Example 1.

[0118] 2. Off-line two-dimensional liquid chromatography system conditions

[0119] 2.1 First dimension hydrophilic interaction chromatography conditions: basically the same as in Example 1, except that the column temperature is 25°C.

[0120] 2.2 Second dimension reversed-phase chromatography conditions: same as in Example 1.

[0121] 3. Mass spectrometry conditions: same as in Example 1.

[0122] Example 3

[0123] The present embodiment provides a method for systematically characterizing the chemical components of the ginseng oral liquid.

[0124] 1. Preparation of sample solution: same as in Example 1.

[0125] 2. Off-line two-dimensional liquid chromatography system conditions

[0126] 2.1 First dimension hydrophilic interaction chromatography conditions: basically the same as in Example 1, except that the column temperature is 45°C.

[0127] 2.2 Second dimension reversed-phase chromatography conditions: same as in Example 1.

[0128] 3. Mass spectrometry conditions: same as in Example 1.

[0129] Example 4

[0130] The present embodiment provides a method for systematically characterizing the chemical components of the ginseng oral liquid.​​​

[0131] 1. Preparation of sample solution: same as Example 1.

[0132] 2. Off-line two-dimensional liquid chromatography system conditions

[0133] 2.1 First dimension hydrophilic interaction chromatography conditions: same as Example 1.

[0134] 2.2 Second dimension reversed-phase chromatography conditions: essentially same as Example 1, except that the chromatographic column was CSH Phenyl-Hexyl.

[0135] 3. Mass spectrometry conditions: same as Example 1.

[0136] Example 5

[0137] The present example provides a method for the systematic characterization of the chemical constituents of a ginseng oral liquid.

[0138] 1. Preparation of sample solution: same as Example 1.

[0139] 2. Off-line two-dimensional liquid chromatography system conditions

[0140] 2.1 First dimension hydrophilic interaction chromatography conditions: same as Example 1.

[0141] 2.2 Second dimension reversed-phase chromatography conditions: essentially same as Example 1, except that the chromatographic column was CSH C18.

[0142] 3. Mass spectrometry conditions: same as Example 1.

[0143] Example 6

[0144] The present example provides a method for the systematic characterization of the chemical constituents of a ginseng oral liquid.

[0145] 1. Preparation of sample solution: same as Example 1.

[0146] 2. Off-line two-dimensional liquid chromatography system conditions

[0147] 2.1 First dimension hydrophilic interaction chromatography conditions: essentially same as Example 1, except that the column temperature was 25°C.

[0148] 2.2 Second dimension reversed-phase chromatography conditions: same as Example 1.

[0149] 3. Mass spectrometry conditions: same as Example 1.

[0150] Example 7

[0151] The present example provides a method for the systematic characterization of the chemical constituents of a ginseng oral liquid.

[0152] 1. Preparation of sample solution: same as Example 1.

[0153] 2. Off-line two-dimensional liquid chromatography system conditions

[0154] 2.1 First dimension hydrophilic interaction chromatography conditions: same as in Example 1, except that the column temperature was 35 °C.

[0155] 2.2 Second dimension reversed-phase chromatography conditions: same as in Example 1.

[0156] 3. Mass spectrometry conditions: same as in Example 1.

[0157] Example 8

[0158] This example provides a method for the systematic characterization of the chemical constituents of Renshen Guben Oral Liquid.

[0159] 1. Preparation of sample solution: same as in Example 1.

[0160] 2. Off-line two-dimensional liquid chromatography system conditions: same as in Example 1.

[0161] 3. Mass spectrometry conditions: same as in Example 1, except that the NCE was set to 10 / 20 / 30 V.

[0162] Example 9

[0163] This example provides a method for the systematic characterization of the chemical constituents of Renshen Guben Oral Liquid.

[0164] 1. Preparation of sample solution: same as in Example 1.

[0165] 2. Off-line two-dimensional liquid chromatography system conditions: same as in Example 1.

[0166] 3. Mass spectrometry conditions: same as in Example 1, except that the NCE was set to 20 / 30 / 40 V.

[0167] Example 10

[0168] This example provides a method for the systematic characterization of the chemical constituents of Renshen Guben Oral Liquid.

[0169] 1. Preparation of sample solution: same as in Example 1.

[0170] 2. Off-line two-dimensional liquid chromatography system conditions: same as in Example 1.

[0171] 3. Mass spectrometry conditions: same as in Example 1, except that the NCE was set to 20 / 40 / 60 V.

[0172] Example 11

[0173] This example provides a network pharmacology analysis of the pharmacologically active ingredients in RSGB related to immune function based on the compounds identified by the two-dimensional liquid chromatography system (2D-LC) of RSGB.

[0174] Compounds identified by RSGB-based two-dimensional liquid chromatography system (2D-LC) were predicted and collected for active ingredients and potential targets using multiple databases including TCMSP, BATMAN-TCM, and SWISS Target Prediction, etc. In addition, disease targets related to immunity were obtained from GeneCard and OMIM databases, focusing on the keyword “immune”. Homo sapiens was used as the research object, and the highest confidence threshold ≥ 0.7 was set for retrieval. Next, protein-protein interaction (PPI) analysis was completed through the STRING website to identify potential targets intersected with disease-related targets. Finally, a traditional Chinese medicine-compound-target network was constructed using Cytoscape3.7.2 software to reflect the relationship between compounds in RSGB and immune-related target genes, as shown in Figure 6 .

[0175] Among the 84 bioactive ingredients and 722 target genes analyzed, 273 common targets were related to immune disorders. Fifteen active ingredients, including dioscin, verbascoside, sucrose, miltirin, 4-hydroxybenzoic acid, Leonurus japonicus sweet, protocatechuic acid, catalpol, gallic acid, acetyl catalpol, paeonol, alisol B, raffinose, trilobatin, and asafetida, had high degree values, indicating that they played an important role in regulating immunity.

[0176] Example 12

[0177] This example adopted a multi-dimensional screening strategy, including network pharmacology analysis results and fingerprint maps of constituent herbs (fingerprint map combination as shown in Figure 7 After evaluation from multiple angles such as effectiveness, safety, quality transferability, and analytical feasibility, a total of 12 Q-Markers were determined, covering 8 traditional Chinese medicines and 8 structural classes of compounds, namely: catalpol, verbascoside, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, loganin, paeonol, paeoniflorin, gallic acid, alisol B, asparagine, and adenosine.

[0178] Example 13

[0179] This example provided a quantitative analysis method for the 12 Q-Markers in Example 5 using UPLC-QQQ-MS.

[0180] 1. Preparation of sample solution

[0181] Ten batches of RSGB samples were diluted 50 times with water, then centrifuged at 20817 g and 4℃ for 10 min. The supernatant after centrifugation was stored in a 4℃ refrigerator for use.

[0182] 1. Method parameters

[0183] Instrument: Waters ACQUITY H-Class UPLC coupled with Xevo TQ-S mass spectrometer; column: BEH Shield RP18 (2.1 x 100 mm, 1.7 μm; Waters), column temperature: 40 °C; mobile phase: 0.1% v / v formic acid in water (A) - acetonitrile (B), flow rate: 0.3 mL / min; injection volume: 3 μL; gradient elution program:

[0184]

[0185] The adjustable multiple reaction monitoring (MRM) mode was used, and the specific parameters were as follows: capillary voltage, +3.0 kV / -2.8 kV; desolvation gas (N2) temperature and flow rate, 500 °C and 1000 L / h, respectively. The key parameters of the MRM method for 12 Q-Markers in RSGB are shown in Table 3.

[0186] Table 3. Key parameters of the MRM method

[0187]

[0188] 2. Method validation

[0189] The results of the method validation under the above chromatographic and mass spectrometric conditions are shown in Figure 8 and Table 4. The 12 Q-markers showed high specificity during the entire analysis process, and other peaks did not have obvious interference with the target compounds. Within a wide concentration range, the peak area of the compounds was positively correlated with their concentration, and the correlation coefficient (R 2 ) was more than 0.995. The LOQ value ranged from 0.17 to 5.68 ng / mL. The relative standard deviations (RSDs) of the intra-day precision, inter-day precision, repeatability, and stability were all less than 10%. These results showed that the method could simultaneously determine the 12 chemical components in the Renshen Guben oral liquid and had high specificity, sensitivity, and accuracy.

[0190] Table 4. Results of method validation for the detection of 12 Q-markers

[0191]

[0192] 3. Determination of the contents of 12 Q-Markers in RSGB

[0193] A total of 10 batches of oral liquid were determined, and the results are shown in Figure 9 Among them, the content of gallic acid was the highest, followed by paeonol, and the content of alisol B was the lowest.

[0194] Comparative Example 1

[0195] This comparative example provides a method for the systematic characterization of the chemical composition of a ginseng oral liquid.

[0196] 1. Preparation of sample solution: same as Example 1.

[0197] 2. Off-line two-dimensional liquid chromatography system conditions

[0198] 2.1 First dimension hydrophilic interaction chromatography conditions: same as Example 1, except that the mobile phase A was water, 10 mM ammonium formate (AF) in water, or 0.1% v / v formic acid (FA) in water.

[0199] 2.2 Second dimension reversed-phase chromatography conditions: same as Example 1.

[0200] Comparative Example 2

[0201] This comparative example provides a method for the systematic characterization of the chemical composition of a ginseng oral liquid.

[0202] 1. Preparation of sample solution: same as Example 1.

[0203] 2. Off-line two-dimensional liquid chromatography system conditions

[0204] 2.1 First dimension hydrophilic interaction chromatography conditions: same as Example 1, except that the column was Xbridge Amide or Xcharge C18.

[0205] 2.2 Second dimension reversed-phase chromatography conditions: same as Example 1.

[0206] Comparative Example 3

[0207] This comparative example provides a method for the systematic characterization of the chemical composition of a ginseng oral liquid.

[0208] 1. Preparation of sample solution: same as Example 1.

[0209] 2. Off-line two-dimensional liquid chromatography system conditions

[0210] 2.1 First dimension hydrophilic interaction chromatography conditions: same as Example 1.

[0211] 2.2 Second dimension reversed-phase chromatography conditions: same as Example 1, except that the mobile phase A was water, 10 mM ammonium formate in water, or 10 mM ammonium acetate (AA) in water.

[0212] Comparative Example 4

[0213] This comparative example provides a method for the systematic characterization of the chemical composition of a ginseng oral liquid.

[0214] 1. Preparation of sample solution: same as Example 1.

[0215] 2 Off-line two-dimensional liquid chromatography system conditions

[0216] 2.1 First dimension hydrophilic interaction chromatography conditions: same as Example 1.

[0217] 2.2 Second dimension reversed-phase chromatography conditions: same as Example 1, except that the chromatographic column was BEH C18 or HSS T3.

[0218] Comparative Example 5

[0219] This comparative example provides a method for the systematic characterization of the chemical constituents of the ginseng Gu Ben oral liquid.

[0220] 1. Preparation of sample solution: same as Example 1.

[0221] 2. Off-line two-dimensional liquid chromatography system conditions: same as Example 1.

[0222] 3. Mass spectrometry conditions: same as Example 1, except that the spray voltage was 3.0 kV or 3.5 kV in positive ion mode and 2.5 kV or 3.5 kV in negative ion mode.

[0223] Comparative Example 6

[0224] This comparative example provides a method for the systematic characterization of the chemical constituents of the ginseng Gu Ben oral liquid.

[0225] 1. Preparation of sample solution: same as Example 1.

[0226] 2. Off-line two-dimensional liquid chromatography system conditions: same as Example 1.

[0227] 3. Mass spectrometry conditions: same as Example 1, except that the capillary temperature was 250, 300, or 400 °C.

[0228] Comparative Example 7

[0229] This comparative example provides a method for the systematic characterization of the chemical constituents of the ginseng Gu Ben oral liquid.

[0230] 1. Preparation of sample solution: same as Example 1.

[0231] 2. Off-line two-dimensional liquid chromatography system conditions: same as Example 1.

[0232] 3. Mass spectrometry conditions: same as Example 1, except that the auxiliary gas temperature was 250, 300, 350, or 450 °C.

[0233] Test Example 1

[0234] This test example investigates the separation of the first dimension chromatography of Examples 1-3.

[0235] As Figure 10As shown, the chromatographic peaks in the first-dimensional chromatography obtained at different column temperatures were well separated. The separation of chromatographic peaks was most obvious when the column temperature was 35℃ (the column temperature of Example 1), especially for compounds with low response that could be detected within 13-16 min.

[0236] Test Example 2

[0237] This test case examines the separation performance of the second-dimensional chromatography in Examples 1, 6, and 7.

[0238] like Figure 11 As shown, the chromatographic peaks in the chromatograms obtained by second-dimensional chromatography at different column temperatures were well separated, with the peak separation effect being relatively better when the column temperature was 45℃ (the column temperature of Example 1).

[0239] Test Example 3

[0240] This test case examines the characteristic fragment ions produced by different compounds in Examples 1, 8-10 under different collision energy settings.

[0241] Nine representative chemical compounds (ginsenoside Rb1, loganin, and paeoniflorin in negative mode; paeonol, tryptophan, and alismazone B in positive mode) were used as monitoring indicators to observe MS2 fragmentation behavior. The results are as follows: Figure 12 As shown, different compound types can generate characteristic fragment ions under different collision energy settings. With increasing collision energy, the abundance of precursor ions decreases, while the abundance of product ions increases. When NCE is set to 10 / 30 / 50V, the number of fragment ions in most compounds increases, improving the accuracy of identifying unknown compounds.

[0242] Test Example 4

[0243] This test case examined the first-dimensional chromatographic peak elution of Example 1 and Comparative Example 1. Figure 13 As shown in the figure. The results indicate that, compared with other aqueous phases, the number of chromatographic peaks detected was significantly higher when 10 mM ammonium acetate (mobile phase A in Example 1) was used as the aqueous phase.

[0244] Test Example 5

[0245] This test example used 22 compounds covering 13 categories of components in RSGB as target analytes. Scatter plots of the relative retention times of the target analytes on the first and second dimensions of the chromatograms of Example 1 and Comparative Example 2 were examined, and the coefficient of determination (R²) of linear regression analysis was used. 2 To assess orthogonality, a scatter plot is used. Figure 14 As shown in Figure A, the 22 compounds exhibited significant differences in relative retention times and good dispersibility on XAmide and XbridgeAmide columns, with their R...2 The values are 0.0012 and 0.0028 respectively, which indicates a high degree of orthogonality. The peak shape and separation efficiency are as shown in Figure 14 As shown in B1 and B2 in FIG. 1, the XAmide column as a one-dimensional column has better peak shape and separation efficiency.

[0246] Test Example 6

[0247] This test example investigates the peak elution of the second dimension chromatography of Example 1, Comparative Example 3. The results are shown in Figure 15 The results show that, compared with other aqueous phases, when water or 0.1% v / v formic acid (mobile phase A of Example 1) is used as the aqueous phase, the number of ion chromatography peaks extracted by the SEIVE software is greater, and the peak shape and response of key marker compounds such as gallic acid and paeonol are better.

[0248] Test Example 7

[0249] This test example investigates the separation selectivity of the second dimension chromatography of Examples 1-3, Comparative Example 4.

[0250] The number of chromatographic peaks, separation degree and the number of full scan primary fragments extracted by the SIEVE software (Thermo Fisher Scientific) in the second dimension chromatogram of Examples 1-3, Comparative Example 4 are compared, and the results are shown in Figure 16 The number of peaks in the second dimension chromatogram of Examples 1-3, Comparative Examples 1, 2 is 6426, 6822, 6585, 5739, 5423 respectively. The results show that the CSH Phenyl-Hexyl, CSH C18 and BEH Shield RP18 columns have greater peak accommodation capacity.

[0251] Test Example 8

[0252] This test example uses nine representative chemical compounds (in negative mode: ginsenoside Rb1, loganin, verbascoside, paeoniflorin, gallic acid; in positive mode: paeonol, hypoxanthine, tryptophan and alisol B) as monitoring indicators to investigate the peak area of the indicator components under different spray voltages in Example 1 and Comparative Example 5. The sample under each condition is in triplicate, and the peak area is normalized and compared.

[0253] The results are shown in Figure 17 The peak area of the five indicator components in positive mode is negatively correlated with the size of the spray voltage; in negative mode, the peak area increases with the increase of the spray voltage, and decreases after a certain degree. Based on the data of each indicator component, the spray voltage of Example 1 can achieve a greater peak area.

[0254] Test Example 9

[0255] The test example takes 9 representative chemical compounds (in negative mode: ginsenoside Rbl, loganin, verbascoside, paeonoside, gallic acid) and in positive mode: paeonol, hypoxanthine, tryptophan and alisol B) as monitoring indicators, and investigates the peak areas of the indicator components of Example 1 and Comparative Example 6 at different capillary temperatures. The samples under each condition are in triplicate, and the peak areas are normalized and compared.

[0256] The results, as shown in Table 1, show that 7 compounds have the strongest response at 350°C (capillary temperature of Example 1). Figure 18

[0257] Test Example 10

[0258] The test example takes 9 representative chemical compounds (in negative mode: ginsenoside Rbl, loganin, verbascoside, paeonoside, gallic acid) and in positive mode: paeonol, hypoxanthine, tryptophan and alisol B) as monitoring indicators, and investigates the peak areas of the indicator components of Example 1 and Comparative Example 7 at different auxiliary gas temperatures. The samples under each condition are in triplicate, and the peak areas are normalized and compared.

[0259] The results, as shown in Table 2, show that 5 compounds have the strongest response at an auxiliary gas temperature of 400°C (auxiliary gas temperature of Example 1). Figure 19

[0260] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.​​

Claims

1. A method for the systematic characterization of the chemical composition of a ginseng oral liquid, characterized by, Chemical components in Renshen Guben oral liquid are detected by offline two-dimensional liquid chromatography coupled with quadrupole high-resolution mass spectrometry; the first dimension chromatography in the offline two-dimensional liquid chromatography is hydrophilic interaction chromatography, and the second dimension chromatography is reversed-phase chromatography.

2. The chemical composition system characterization method of claim 1, wherein, The chromatographic conditions of the first dimension chromatography are as follows: The chromatographic column is an octadecylsilane-bonded silica gel chromatographic column; The mobile phase A is 9-11 mM ammonium acetate aqueous solution, and the mobile phase B is acetonitrile; the linear gradient elution program is as follows: The flow rate is 1.0 mL / min; From 0 to 27 min, the eluent is collected as a fraction every 3 min, and the eluent from 27 to 32 min is collected as a fraction; after drying, each fraction is dissolved in 50% v / v methanol, centrifuged, and the supernatant is used for second dimension chromatography analysis; The chromatographic conditions of the second dimension chromatography are as follows: The chromatographic column is a reversed-phase chromatographic column; The mobile phase A is 0.1% v / v formic acid aqueous solution, and the mobile phase B is acetonitrile; The gradient elution program is as follows: The flow rate is 0.3 mL / min.

3. The chemical composition system characterization method of claim 2, wherein, In the chromatographic conditions of the first dimension chromatography, the chromatographic column is Acchrom XAmide with a specification of 4.6*150 mm and a particle size of 5 μm; and / or In the chromatographic conditions of the first dimension chromatography, the mobile phase A is 10 mM ammonium acetate aqueous solution; and / or In the chromatographic conditions of the first dimension chromatography, the column temperature is 25-45 °C; and / or In the chromatographic conditions of the second dimension chromatography, the chromatographic column is CSH Phenyl-Hexyl, CSH C18 and BEH Shield RP18 with a specification of 2.1*100 mm and a particle size of 1.7 μm; and / or In the chromatographic conditions of the second dimension chromatography, the column temperature is 25-45 °C.

4. The chemical composition system characterization method of claim 3, wherein, In the chromatographic conditions of the first dimension chromatography, the column temperature is 35 °C; and / or In the chromatographic conditions of the second dimension chromatography, the chromatographic column is BEH Shield RP18; and / or In the chromatographic conditions of the second dimension chromatography, the column temperature is 45 °C.

5. The method of chemical composition system characterization according to any one of claims 1 to 4, characterized in that, The quadrupole high-resolution mass spectrometer is Exploris 120 Q-Orbitrap mass spectrometer equipped with HESI source, and data are collected in positive and negative ion switching mode.

6. The chemical composition system characterization method of claim 5, wherein, The parameters of the mass spectrometer are as follows: spray voltage, -3.0 kV / +2.5 kV; sheath gas pressure, 35 arb; auxiliary gas pressure, 10 arb; sweep gas pressure, 0 arb; capillary temperature, 350 °C; Temperature of auxiliary gas, 400°C; resolution of primary and secondary 60,000, 15,000, respectively; scan range m / z 100-1500, AGC set at 1 x 10 6 and 1 x 10 5 ; MS 1 Top 4 ions with the highest intensity automatically selected to trigger high-energy collision-induced dissociation MS 2 Fragmentation; normalized collision energy set to 10-20 / 20-40 / 30-60 V, isolation width of 4.0 Da; dynamic exclusion time set to 6 S.

7. The use of the chemical composition system characterization method according to any one of claims 1-6 in screening quality markers of Renshen Guuben Oral Liquid, characterized in that, By network pharmacology, active ingredients related to immune function are screened from the compounds identified by the chemical component system characterization method, and quality markers are further screened in combination with the fingerprint of the traditional Chinese medicine components of Renshen Guben oral liquid.

8. A set of quality markers of ginseng GuBen oral liquid, characterized in that, Comprise: Albizziol, Verbascoside, Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Re, Loganin, Paeonol, Paeoniflorin, Gallic Acid, Alisol B, Asparagine and Adenosine.

9. The method of detecting the quality marker of claim 8, characterized in that, The quality markers are analyzed by liquid chromatography-mass spectrometry; The liquid chromatography conditions in the liquid chromatography-mass spectrometry are as follows: The chromatographic column is an octadecylsilane-bonded silica gel chromatographic column; The mobile phase A is 0.1% v / v formic acid aqueous solution, and the mobile phase B is acetonitrile; the linear gradient elution program is as follows: The flow rate is 0.3 mL / min; The mass spectrometer in the liquid chromatography-mass spectrometry adopts an adjustable multiple reaction monitoring mode.

10. The detection method according to claim 9, characterized in that, The chromatographic column is a BEH Shield RP18 with a specification of 2.1*100 mm, 1.7 μm; and / or The column temperature is 35-45 DEG C; and / or The mass spectrometer in the liquid chromatography-mass spectrometry adopts an Xevo TQ-S mass spectrometer; and / or The mass spectrometer sets the parent ion, daughter ion, orifice voltage, collision energy and acquisition mode for each mass marker as:

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