Fingerprint of Qilongzhuanger Oral Liquid and Its Application in Quality Control

Abstract

The present invention relates to a fingerprint of Qilong Zhuang'er oral liquid and an application thereof in quality control. The fingerprint of the Qilong Zhuang'er oral liquid in positive ion mode is substantially consistent with FIG1 or FIG3B, and 24 common peaks are determined. The fingerprint in negative ion mode is substantially consistent with FIG2 or FIG3D, and 17 common peaks are determined.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine analysis and quality control, and particularly relates to a fingerprint spectrum of Qilong Zhuanger oral liquid and an application thereof in quality control. Background Art

[0002] Qilong Zhuang'er Oral Liquid is a Class A OTC Chinese patent medicine formulated by renowned pediatrician Dr. Wu Xueqiao based on traditional Chinese medicine theory. It is designed to treat malnutrition, five delayed and five soft symptoms, and blood deficiency caused by spleen and kidney deficiency in children. It uses Astragalus and Codonopsis as the main ingredients, Rehmannia glutinosa, Angelica sinensis, donkey-hide gelatin, deer antler glue, and tortoise shell glue as the auxiliary ingredients, and Dragon Bone, Oyster, Atractylodes macrocephala, Chinese Yam, Chicken's Gizzard Stone, Crataegus, and Malt as the adjuvant ingredients. The whole formula strengthens the spleen and kidneys, replenishes Qi and blood, replenishes essence and marrow, aids digestion, and soothes the stomach. It is primarily used clinically to treat rickets in children. The current quality control method for Qilong Zhuang'er Oral Liquid is to determine the content of astragaloside IV by thin-layer scanning. [1] , it is difficult to fully reflect and control the preparation quality of Qilong Zhuang'er oral liquid. The chemical composition of traditional Chinese medicine and compound preparations is complex, and quality control is a difficult point in their research. Detecting one component cannot represent the overall quality. The present invention uses UHPLC-Q-Exactive Orbitrap MS technology to simultaneously detect positive and negative ion compounds, and establishes fingerprint spectra of Qilong Zhuang'er oral liquid in positive and negative ion modes. At the same time, 10 batches of samples are evaluated for similarity, and the common peaks are identified by mass spectrometry. The chemical composition fingerprint characteristics of the preparation are more comprehensively characterized from the positive and negative dimensions, providing a scientific basis for the comprehensive quality evaluation and pharmacological substance research of Qilong Zhuang'er oral liquid. Summary of the Invention

[0003] The present invention provides a fingerprint spectrum of Qilongzhuanger oral liquid, which is characterized in that when the chromatographic conditions and mass spectrometry conditions are as follows, the fingerprint spectrum of the Qilongzhuanger oral liquid in positive ion mode is substantially the same as Figure 1 or Figure 3 B, the fingerprint spectrum in negative ion mode is basically the same as Figure 2 or Figure 3 D consistent;

[0004] The chromatographic conditions are as follows:

[0005] The chromatographic column was a Thermo Accucore aQ RP18, 2.1 mm × 150 mm, 2.6 μm. The mobile phases were methanol as phase A and 0.1% formic acid in water as phase B. The gradient elution was as follows: 0% A from 0 to 5 min; 0% to 58% A from 5 to 45 min; and 58% to 65% A from 45 to 60 min. The column temperature was 25°C, the flow rate was 0.3 ml / min, and the injection volume was 2 μl.

[0006] Mass spectrometry conditions are as follows:

[0007] Thermo UHPLC-Q-Exactive Focus liquid chromatography-mass spectrometry (LC-MS) was used, with a heated electrospray ionization (HESI) source and simultaneous positive and negative ion detection modes. The spray voltage was set to 2800 V in negative ion mode and 3500 V in positive ion mode. The collision voltage was set to step energies of 20, 40, and 60 eV. The scanning mode was full scan / data-dependent secondary mass spectrometry (full MS / dd-MS). 2 ), Full MS resolution is 70000, dd-MS 2 The resolution was 17500, the scan range was m / z 80-1200, the ion transfer capillary temperature was 320°C, the lens voltage was 55 kPa, the spray chamber temperature was 350°C, and the sheath and auxiliary gas flow rates were 40 arb and 10 arb, respectively.

[0008] Another embodiment of the present invention provides a fingerprint of the Qilong Zhuanger oral solution, characterized in that the fingerprint of the Qilong Zhuanger oral solution in positive ion mode is substantially the same as that of the Qilong Zhuanger oral solution. Figure 1 or Figure 3 B, with 24 characteristic fingerprint peaks, including peak 4 for adenosine, peak 5 for guanosine, peak 7 for L-tryptophan, peak 12 for calycosin, peak 17 formononetin, peak 18 for calycosin, peak 21 for 3-hydroxy-9,10-dimethoxypterocarpa, peak 23 formononetin, and peak 24 for atractylodes lactone III. Peak 1 for isoleucine, peak 3 for hordenine, peak 19 for ligustrin F, and peak 22 for 7,2'-dihydroxy-3',4'-dimethoxyisoflavanone.

[0009] Another embodiment of the present invention provides a fingerprint of the Qilong Zhuanger oral solution, characterized in that the fingerprint of the Qilong Zhuanger oral solution in negative ion mode is substantially the same as that of the Qilong Zhuanger oral solution. Figure 2 or Figure 3 D, with 17 characteristic fingerprint peaks, including peak 1 for pyroglutamic acid, peak 2 for uridine, peak 7 for caffeic acid, peak 9 for calycosin, peak 10 for isocalycoside, peak 11 for codonopsis pilosula, peak 12 formononetin, peak 14 for calycosin, and peak 16 formononetin. Peak 3 for monomilite glycoside, peak 5 for leonurin, and peak 15 for ligustrin F.

[0010] Another embodiment of the present invention provides the use of the fingerprint of the Qilong Zhuang'er oral liquid in the quality control and component analysis of the Qilong Zhuang'er oral liquid. The fingerprint is selected from the fingerprint in positive ion mode and the fingerprint in negative ion mode.

[0011] Another embodiment of the present invention provides an application of the fingerprint of the Qilong Zhuang'er oral liquid in the quality control of the Qilong Zhuang'er oral liquid, characterized in that the application comprises the following steps:

[0012] (1) Preparation of test solution: accurately measure 2 ml of Qilong Zhuang'er oral solution and place it in a 15 ml centrifuge tube. Add 8 ml of acetonitrile and vortex to mix for 5 min. After standing at 4°C for 0.5 h, centrifuge at 2500 rpm for 20 min. Take 1 ml of the supernatant and dry the solvent. Dissolve the residue in 30% methanol-water and dilute to 1 ml. Shake and filter through a 0.22 μm microporous filter.

[0013] (2) Take the test solution obtained in step (1), and inject and detect the sample under the following chromatographic conditions and mass spectrometry conditions to obtain the spectrum of the test sample;

[0014] Chromatographic conditions were as follows: Thermo Accucore aQ RP18 column, dimensions: 2.1 mm × 150 mm, 2.6 μm; mobile phase: methanol as phase A, 0.1% formic acid in water as phase B; gradient elution: 0% A from 0 to 5 min; 0% to 58% A from 5 to 45 min; 58% to 65% A from 45 to 60 min; column temperature: 25°C, flow rate: 0.3 ml / min, injection volume: 2 μl;

[0015] The mass spectrometry conditions were as follows: a Thermo UHPLC-Q-Exactive Focus liquid chromatography-mass spectrometry instrument was used, with a heated electrospray ionization (HESI) source, simultaneous positive and negative ion detection mode, a spray voltage of 2800 V in negative ion mode, and a spray voltage of 3500 V in positive ion mode; collision voltages were set at step energies of 20, 40, and 60 eV; and a full-scan primary / data-dependent secondary mass spectrometry (dd-MS) scanning mode. 2 ), Full MS resolution is 70000, dd-MS 2 The resolution was 17500, the scan range was m / z 80–1200, the ion transfer capillary temperature was 320°C, the lens voltage was 55 kPa, the spray chamber temperature was 350°C, and the sheath and auxiliary gas flow rates were 40 arb and 10 arb, respectively;

[0016] (3) Compare the fingerprint of the sample obtained in step (2) with the fingerprint of the Qilong Zhuang'er oral liquid described in the present invention. The Qilong Zhuang'er oral liquid with a similarity of more than 0.96 is a qualified product.

[0017] Compared with the prior art, the advantages of the present invention are: (1) the present invention makes up for the shortcomings of the prior art, and uses UHPLC-Q-Exactive Orbitrap MS technology to establish fingerprints of Qilong Zhuang'er oral liquid in positive ion mode and negative ion mode, providing an effective method for quality control of Qilong Zhuang'er oral liquid; (2) the present invention performs full spectrum peak matching on 10 batches of Qilong Zhuang'er oral liquid, adopts multi-point calibration mode, and generates positive ion mode fingerprints by averaging method. Figure 1 or Figure 3 B, 24 common peaks were determined; the fingerprint in negative ion mode is shown in Figure 2 or Figure 3 D, a total of 17 peaks were determined; (3) Qilongzhuang'er oral liquid contains glue-like medicinal materials rich in amino acids, such as donkey-hide gelatin, deer horn glue, and tortoise shell glue. Amino acid compounds cannot be directly detected by chromatographic peaks under conventional HPLC-UV conditions, and the UV detector can only detect compounds containing ultraviolet luminescent groups, which has certain limitations. The mass spectrometer is a general-purpose detector that has a good response signal for amino acids and other compounds that do not absorb ultraviolet light. Therefore, this study used UHPLC-Q-Exactive Orbitrap MS technology for the first time to establish two fingerprints of Qilongzhuang'er oral liquid in positive and negative ion detection modes. The fingerprints are rich in information and have good separation effects. They comprehensively reflect the component information of Qilongzhuang'er oral liquid from two different dimensions, positive and negative. In addition, high-resolution mass spectrometry can directly use the data information collected to identify the compounds. This has certain reference significance for establishing a scientific and reasonable quality evaluation method for Qilongzhuang'er oral liquid and improving its quality control standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the fingerprints of 10 batches of Qilong Zhuanger oral liquid in positive ion mode.

[0019] Figure 2 These are the fingerprints of 10 batches of Qilong Zhuanger oral liquid in negative ion mode.

[0020] Figure 3 These are the total ion current base peak graphs of the mixed reference solution (A: positive ion mode, C: negative ion mode) and the control fingerprint of Qilong Zhuang'er oral liquid (B: positive ion mode, D: negative ion mode); A and B (positive ion mode): 4. adenosine; 5. guanosine; 7. L-tryptophan; 12. calycosin; 17. formononetin; 18. calycosin; 21. 3-hydroxy-9,10-dimethoxypterocarpa; 23. formononetin; 24. atractylodes lactone III; C and D (negative ion mode): 1. pyroglutamic acid; 2. uridine; 7. caffeic acid; 9. calycosin; 10. isocalycoside; 11. codonopsis pilosula; 12. formononetin; 14. calycosin; 16. formononetin.

[0021] Figure 4 This is a diagram of the mass spectrometry fragmentation pathway of isocarpus glycosides. DETAILED DESCRIPTION

[0022] 1. Instruments and Materials

[0023] The instrumentation system consisted of an UltiMate 3000 series high-performance liquid chromatograph (Dionex, USA), a ThermoFisher Q Exactive Focus mass spectrometer equipped with a heated electrospray ionization (HESI) source, and Xcalibur 4.0 data processing software. A KQ-400DE ultrasonic cleaner was used (Kunshan Ultrasonic Instrument Co., Ltd.), along with a BT25S 1 / 100,000 electronic analytical balance and a BS210S 1 / 10,000 electronic analytical balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0024] Materials: 10 batches of Qilongzhuanger oral solution were provided by Xi'an Zhengda Pharmaceutical Co., Ltd. (specification: 10 ml / tube; batch numbers: 211135 (S1), 211136 (S2), 211237 (S3), 211238 (S4), 211239 (S5), 211240 (S6), 211241 (S7), 2111242 (S8), 211243 (S9), 211244 (S10). 9), 211244 (S10). Adenosine (lot number MUST-21070613), guanosine (lot number MUST-22021408), and isocalycoside (lot number MUST-22030810) were purchased from Chengdu Mansite Biotechnology Co., Ltd.; L-tryptophan (lot number wkq22011707), isoflavone glycosides (lot number wkq20010904), and codonopsis pilosula Pantothenoside (lot number: wkq17110203), formononetin (lot number: wkq20060507), calycosin (lot number: wkq20010906), and formononetin (lot number: wkq20062208) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; 3-hydroxy-9,10-dimethoxypterotanane (lot number: HR2182W1), atractylodes lactone III (lot number: HS20913B1), pyroglutamic acid (lot number: HR22271W2), and uridine (lot number: HS2175B1) were purchased from Baoji Chenguang Biotechnology Co., Ltd.; and caffeic acid (lot number: 17122804) was purchased from Chengdu Pufeide Biotechnology Co., Ltd. All reference substances had a purity of ≥98%. Methanol and formic acid were chromatographically grade (Fisher Reagent Company, USA); water was distilled water; all other reagents were of analytical grade.

[0025] 2 Methods and Results

[0026] 2.1 Chromatographic conditions

[0027] The chromatographic column was a Thermo Accucore aQ RP18 (2.1 mm × 150 mm, 2.6 μm); the mobile phase was methanol (A)-0.1% formic acid aqueous solution (B) with gradient elution (0–5 min, 0% A; 5–45 min, 0%–58% A; 45–60 min, 58%–65% A); the column temperature was 25°C, the flow rate was 0.3 ml / min, and the injection volume was 2 μl.

[0028] 2.2 Mass spectrometry conditions

[0029] Thermo UHPLC-Q-Exactive Focus liquid chromatography-mass spectrometry instrument was used. A heated electrospray ionization (HESI) source was used, with simultaneous positive and negative ion detection modes. The spray voltage was set to 2800 V in negative ion mode and 3500 V in positive ion mode. The collision voltage was set to a step energy of 20, 40, and 60 eV. The scanning mode was full scan / data-dependent secondary mass spectrometry (full MS / dd-MS). 2 ), Full MS resolution is 70000, dd-MS 2 The resolution was 17500, the scan range was m / z 80-1200, the ion transfer capillary temperature was 320°C, the lens voltage was 55 kPa, the spray chamber temperature was 350°C, and the sheath and auxiliary gas flow rates were 40 arb and 10 arb, respectively.

[0030] 2.3 Preparation of sample solution

[0031] 2.3.1 Preparation of Reference Solution Two sets of mixed reference solutions were prepared for positive and negative detection modes. The reference concentrations were 65 μg / ml of pyroglutamic acid, 70 μg / ml of uridine, 86 μg / ml of adenosine, 37 μg / ml of guanosine, 15 μg / ml of L-tryptophan, 20 μg / ml of caffeic acid, 144 μg / ml of calycosin, 45 μg / ml of isocalycoside, 24 μg / ml of codonopsis pilosulosin, 46 μg / ml of formononetin, 55 μg / ml of calycosin, 18 μg / ml of 3-hydroxy-9,10-dimethoxypterotanane, 25 μg / ml of formononetin, and 19 μg / ml of atractylodes lactone III.

[0032] 2.3.2 Preparation of test solution: Accurately measure 2 ml of Qilong Zhuang'er oral liquid, place it in a 15 ml centrifuge tube, add 8 ml of acetonitrile, oscillate and mix on a vortex oscillator for 5 min, let it stand at 4 °C for 0.5 h, centrifuge at 2500 rpm for 20 min, take 1 ml of the supernatant, blow dry the solvent, dissolve the residue in 30% methanol-water and make up to 1 ml, shake, and filter through a 0.22 μm microporous filter membrane.

[0033] 2.3.3 Preparation of single herb samples Take samples according to the corresponding prescription quantity and ratio of each single herb in the prescription to prepare single herb sample solutions.

[0034] 2.4 Fingerprint Methodology Review

[0035] 2.4.1 Precision Test: Sample solution from batch number 211135 was injected six times in succession. In positive ion mode, common peak 18, calycosin, was used as the reference peak, and in negative ion mode, common peak 9, calycosin, was used as the reference peak. The relative standard deviations (RSDs) of the relative retention times and peak areas of the 24 common peaks in positive ion mode and the 17 common peaks in negative ion mode were calculated relative to the selected reference peaks. The RSDs were less than 0.62% and 2.84%, respectively, indicating that the method has good precision.

[0036] 2.4.2 Repeatability Test: Six replicates of 2 mL of Qilong Zhuang'er Oral Solution (Batch No. 211135) were prepared and analyzed in parallel using the method described in "2.3.2." The RSDs of the relative retention times and relative peak areas of the 24 common peaks in positive ion mode and the 17 common peaks in negative ion mode relative to selected reference peaks were calculated. The results were less than 0.55% and 2.97%, respectively, demonstrating good repeatability of this method.

[0037] 2.4.3 Stability Test: 2 mL of Qilong Zhuang'er Oral Solution (Batch No. 211135) was prepared as a test solution according to the method described in "2.3.2." The sample was then placed at room temperature for analysis at 0, 2, 4, 8, 12, and 24 injections. The relative retention times and peak areas of the 24 common peaks in positive ion mode and the 17 common peaks in negative ion mode relative to the selected reference peaks were calculated. The RSDs were less than 0.89% and 3.16%, respectively. This indicates that the sample solution exhibited good stability after 24 hours at room temperature.

[0038] 2.5 Fingerprint establishment and similarity evaluation

[0039] 2.5.1 Fingerprint establishment and similarity analysis of 10 batches of samples. Ten batches of Qilong Zhuanger oral liquid (S1-S10) were taken and the sample solutions were prepared according to the method under "2.3.2". The chromatographic conditions and mass spectrometry conditions under "2.1" and "2.2" were used for sample injection and analysis. The data files were exported to "txt" format using Xcalibar 4.0 software. The 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation software was used for full spectrum peak matching. The multi-point calibration mode and the average method (time window 0.1 min) were used to generate the positive ion mode fingerprint. Figure 1 , a total of 24 peaks were determined; the fingerprint of negative ion mode is shown in Figure 2, 17 common peaks were identified. In the positive ion mode, the similarities between S1-S10 and the control fingerprint were 0.995, 0.995, 0.995, 0.993, 0.997, 0.986, 0.989, 0.993, 0.994, and 0.986, respectively. In the negative ion mode, the similarities between S1-S10 and the control fingerprint were 0.997, 0.991, 0.997, 0.997, 0.997, 0.994, 0.968, 0.973, 0.988, and 0.993, respectively. The results showed that the similarities between the 10 batches of samples in both positive and negative ion detection modes were greater than 0.96, indicating good consistency between different batches of Qilong Zhuang'er Oral Liquid, and the quality of the raw materials and the production process were relatively stable. 2.5.2 Attribution of Common Peaks in Fingerprints. Qilong Zhuang'er Oral Solution test solution, individual medicinal herbs, and blank solvent were injected according to the chromatographic and mass spectrometry conditions described in "2.1" and "2.2." The total ion current base peak of the mass spectrometer was recorded. The peaks in the chromatogram were compared, and the major chromatographic peaks were assigned to the medicinal herbs. The results are shown in Tables 1 and 2.

[0040] 2.5.3 Identification of common peaks in fingerprints Based on the primary and secondary data of high-resolution mass spectrometry and references, a total of 24 common peaks in positive ion mode and 17 common peaks in negative ion mode were identified. The results are shown in Tables 1 and 2. Among them, 9 compounds in positive ion mode were identified by comparison with reference substances as adenosine (common fingerprint peak No. 4), guanosine (common fingerprint peak No. 5), L-tryptophan (common fingerprint peak No. 7), calycosin (common fingerprint peak No. 12), formononetin (common fingerprint peak No. 17), calycosin (common fingerprint peak No. 18), 3-hydroxy-9,10-dimethoxypterocarpone (common fingerprint peak No. 21), formononetin (common fingerprint peak No. 23), and atractylodes lactone III (common fingerprint peak No. 24). Figure 3 A; In negative ion mode, 9 compounds were identified by comparison with reference substances as pyroglutamic acid (common fingerprint peak No. 1), uridine (common fingerprint peak No. 2), caffeic acid (common fingerprint peak No. 7), calycosin (common fingerprint peak No. 9), isocalycoside (common fingerprint peak No. 10), codonopsis pilosula (common fingerprint peak No. 11), formononetin (common fingerprint peak No. 12), calycosin (common fingerprint peak No. 14), formononetin (common fingerprint peak No. 16), see Figure 3 C. The mass spectrometry identification process of the common peak is illustrated using isocarpus glycoside as an example. The quasi-ion peak of isocarpus glycoside in negative ion mode is m / z 623.1981[MH]-, and its molecular formula is C after fitting with Xcalibar 4.0 software. 29 H 36 O 15 The secondary fragment ion is m / z 461.1671[MH-C9H6O3] -and m / z 161.0244 [MHC 20 H 30 O 12 ] - , produced by the cleavage of the caffeoyl bond, m / z 461.1671 [MH-C9H6O3] - Further loss of one molecule of rhamnose produces the fragment m / z 315.1079 [MH-C9H6O3-Rha] - ,m / z 161.0244[MHC 20 H 30 O 12 ] - The loss of one molecule of CO produces a fragment ion m / z 133.0295 [MHC 20 H 30 O 12 -CO] - According to the above cleavage pathway, refer to relevant literature [5] The compound 10 in negative ion mode was confirmed to be isocarpus glycoside by comparison with reference substances. Its possible mass spectrometry fragmentation pathway is shown in Figure 4 .

[0041]

[0042]

[0043] References

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Claims

1. A method for constructing a fingerprint of Qilong Zhuanger oral liquid, characterized in that: The steps include: Ten batches of Qilongzhuang'er oral liquid were prepared as follows, and sample solutions were injected and analyzed according to the following chromatographic and mass spectrometric conditions. The data files were exported to txt format using Xcalibar 4.0 software. Full-spectrum peak matching was performed using the 2012 version of Chinese medicine chromatographic fingerprint similarity evaluation software. Multi-point calibration mode, averaging method, and a time window of 0.1 min were used to generate fingerprints in positive ion mode, identifying 24 common peaks; and fingerprints in negative ion mode, identifying 17 common peaks. The method for preparing the sample solution, including the preparation of the reference solution and the test solution, was as follows: (1) Preparation of reference solution: Two sets of mixed reference solutions were prepared in positive and negative detection modes. The reference solution contained pyroglutamic acid, uridine, adenosine, guanosine, L-tryptophan, caffeic acid, calycosin, isocalycoside, codonopsis pilosula, formononetin, calycosin, 3-hydroxy-9,10-dimethoxypterotanane, formononetin and atractylodes lactone III. (2) Preparation of test solution: accurately measure 2 ml of Qilongzhuanger oral solution and place it in a 15 ml centrifuge tube. Add 8 ml of acetonitrile and vortex to mix for 5 min. After standing at 4 °C for 0.5 h, centrifuge at 2500 rpm for 20 min. Take 1 ml of the supernatant and dry the solvent. Dissolve the residue in 30% methanol-water and make up to 1 ml. Shake and filter through a 0.22 μm microporous filter membrane. Chromatographic conditions were as follows: the column was Thermo Accucore aQ RP18, specifications: 2.1 mm × 150 mm, 2.6 μm; Mobile phase: methanol as phase A, 0.1% formic acid aqueous solution as phase B; gradient elution: 0-5 min, 0% A; 5-45 min, 0%-58% A; 45-60 min, 58%-65% A; The column temperature was 25 °C, the volume flow rate was 0.3 ml / min, and the injection volume was 2 μl; The mass spectrometry conditions were as follows: a Thermo UHPLC-Q-Exactive Focus liquid chromatography-mass spectrometer was used, with a heated electrospray ionization (HESI) source, simultaneous positive and negative ion detection mode, a spray voltage of 2800 V in negative ion mode, and a spray voltage of 3500 V in positive ion mode; collision voltages were set at step energies of 20, 40, and 60 eV; and a full-scan primary / data-dependent secondary mass spectrometry (dd-MS) scanning mode. 2 ), Full MS resolution of 70000, dd-MS 2 The resolution was 17500, the scan range was m / z 80–1200, the ion transfer capillary temperature was 320°C, the lens voltage was 55 kPa, the nebulizer temperature was 350°C, and the sheath and auxiliary gas flow rates were 40 arb and 10 arb, respectively.

2. The construction method according to claim 1, characterized in that The fingerprint spectrum of the Qilong Zhuang'er oral liquid in positive ion mode has 24 characteristic fingerprint chromatographic peaks, among which peak 4 is adenosine, peak 5 is guanosine, peak 7 is L-tryptophan, peak 12 is calycosin, peak 17 is formononetin, peak 18 is calycosin, peak 21 is 3-hydroxy-9,10-dimethoxypterocarpa, peak 23 is formononetin, and peak 24 is atractylodes lactone III.

3. The construction method according to claim 2, characterized in that Peak 1 is isoleucine, peak 3 is hordenine, peak 19 is ligustolactone F, and peak 22 is 7,2'-dihydroxy-3',4'-dimethoxyisoflavanone.

4. The construction method according to claim 1, characterized in that The fingerprint spectrum of the Qilong Zhuang'er oral liquid in negative ion mode has 17 characteristic fingerprint chromatographic peaks, among which peak 1 is pyroglutamic acid, peak 2 is uridine, peak 7 is caffeic acid, peak 9 is calycosin, peak 10 is isocalycoside, peak 11 is codonopsis pilosula, peak 12 is formononetin, peak 14 is calycosin, and peak 16 is formononetin.

5. The construction method according to claim 4, characterized in that Peak 3 is monomilite glycoside, peak 5 is leonurin, and peak 15 is ligustrin F.

6. Application of the fingerprint of Qilong Zhuang'er oral liquid constructed according to the construction method according to any one of claims 1 to 5 in the component analysis of Qilong Zhuang'er oral liquid.

7. The use according to claim 6, characterized in that The fingerprint spectrum is selected from the fingerprint spectrum under positive ion mode and the fingerprint spectrum under negative ion mode.

8. Application of the fingerprint of Qilong Zhuang'er oral liquid constructed according to the construction method according to any one of claims 1 to 5 in the quality control of Qilong Zhuang'er oral liquid, characterized in that: The application comprises the following steps: (1) Preparation of test solution: accurately measure 2 ml of Qilongzhuanger oral solution and place it in a 15 ml centrifuge tube. Add 8 ml of acetonitrile and vortex to mix for 5 min. After standing at 4 °C for 0.5 h, centrifuge at 2500 rpm for 20 min. Take 1 ml of the supernatant and dry the solvent. Dissolve the residue in 30% methanol-water and make up to 1 ml. Shake and filter through a 0.22 μm microporous filter. (2) taking the test solution obtained in step (1), injecting the sample and detecting the spectrum of the test sample under the chromatographic conditions and mass spectrometry conditions described in claim 1; (3) Compare the fingerprint of the sample obtained in step (2) with the fingerprint of the Qilong Zhuang'er oral liquid constructed according to any one of claims 1 to 5. The Qilong Zhuang'er oral liquid with a similarity of more than 0.96 is a qualified product.

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