A method for detecting non-volatile chemical components in Xingnaojing injection

Abstract

The invention relates to a method for detecting non-volatile chemical components in Xingnaojing injection. The method adopts an optimized ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap mass spectrometry method, wherein the liquid phase conditions are as follows: a chromatographic column is Waters BEH C18, a mobile phase flow rate is set to 0.2 mL / min, a column temperature is 30 DEG C, and the method is used to separate biological samples. The mobile phase consists of (A) 0.1% formic acid solution and (B) acetonitrile. The gradient elution re-equilibrium time is 10 min, and the injection volume is 15 μL. The method analyzes the non-volatile components in Xingnaojing injection and identifies the components using a reference substance.

Description

Technical Field

[0001] The invention relates to a method for determining the chemical components of Xingnaojing injection, and specifically adopts an ultra high performance liquid chromatography-quadrupole-electrostatic field orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) method to detect and analyze the non-volatile components in Xingnaojing injection. Background Art

[0002] The unclear pharmacological substances and mechanisms of action of traditional Chinese medicine have always been a key issue restricting the modernization of traditional Chinese medicine. Our research group previously proposed the concept of "manifested effect form" and the hypothesis of "manifested effect theory" of traditional Chinese medicine, believing that the "manifested effect form" of traditional Chinese medicine after entering the body includes the original ingredients and their metabolites, and the "superposition effect" and "synergistic effect" of many ingredients and many metabolites together constitute the important connotation of the mechanism of action of traditional Chinese medicine. Therefore, it is of great significance to clarify the original ingredients and metabolites of traditional Chinese medicine entering the body and screen out the active compounds in the body for the study of pharmacological substances of traditional Chinese medicine.

[0003] Xingnaojing injection is a modified version of the ancient Angong Niuhuang Pill. It is made from musk, borneol, gardenia, and turmeric extracted by two steam distillations. It has the effects of awakening the mind, clearing away heat and detoxifying, cooling blood and activating blood circulation. It is clinically used to treat cerebral infarction and cerebral hemorrhage. So, what are the chemical components in Xingnaojing injection? What metabolites will these chemical components produce after entering the body? Do these original components and metabolites have pharmacological activity against ischemic stroke? Clarifying the above questions is very important for revealing the pharmacological substances of Xingnaojing injection.

[0004] Since there are some non-volatile compounds in Xingnaojing injection, such as curcuminol, germacron, curcumadione and curcumol, the ultra-high performance liquid chromatography-quadrupole-exactive field orbitrap mass spectrometry (UHPLC-Q-ExactiveOrbitrap MS) method can efficiently and sensitively detect the non-volatile components in Xingnaojing injection, and the method is simple to operate. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an ultra high performance liquid chromatography-quadrupole-electrostatic field orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) method for detecting non-volatile components in Xingnaojing injection.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a method for determining the non-volatile chemical components in Xingnaojing injection based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) technology, the steps of which are as follows:

[0007] Weigh curcumenol, germacrone, curcumadione and curcumol, add appropriate amount of methanol to dissolve, and obtain standard solution. Inject the standard solution and Xingnaojing injection into the chromatograph according to the following method to obtain the chromatogram, and perform LC-MS analysis.

[0008] Liquid phase conditions:

[0009] The chromatographic column was Waters BEH C18 (100×2.1 mm, 1.7 μm).

[0010] The mobile phase flow rate was set at 0.2 mL / min and the column temperature was set at 30 °C for separation of biological samples.

[0011] The mobile phase consisted of (A) 0.1% formic acid solution and (B) acetonitrile.

[0012] The mobile phase gradient was: 5% (v / v) B from 0 to 5 min, 5% to 30% B from 5 to 45 min, 30% to 60% B from 45 to 46 min, 60% to 60% B from 46 to 50 min, 60% to 100% B from 50 to 52 min, maintained at 100% B for 8 min, reduced to 5% B from 60 to 60.1 min, and the gradient elution re-equilibrium time was 10 min.

[0013] The injection volume was 15 μL.

[0014] Mass spectrometry conditions:

[0015] Scan mode: Full MS / dd-MS 2 .

[0016] In Full MS mode, the resolution is 140000; the AGC target is 1×10 6 , Maximum IT is 100 ms, and the scanning range is m / z 53.4-800.

[0017] The dd-MS2 / dd-SIM parameters are set to: resolution 17500, AGC target 1×10 5 , Maximum IT is 50ms, loop count is 10, isolation window is m / z 2; CE is 15, 25, 35eV. The ionization mode is ESI positive ion scanning, the ion source is HESI source, the ion source spray voltage is 3.0kV positive ion, the capillary temperature is 320℃, the sheath gas flow rate is 40 units N2, the auxiliary gas flow rate is 10 units N2, and the S-Lens RF level is 55.

[0018] Through the chromatogram, the chromatographic peaks of the components curcumenol, germacrone, curcumadione and curcumol in the Xingnaojing injection are analyzed and obtained, and the contents of these components in the Xingnaojing injection can be calculated through the chromatographic peaks.

[0019] The current quality standard for Xingnaojing injection is the national drug standard WS3-B-3353-98-2003, which uses gas chromatography to control the content of dextrorotatory borneol in Xingnaojing injection (not less than 0.7g / L) as an indicator, and to qualitatively characterize musk ketone. The current standard cannot distinguish between synthetic borneol, natural borneol, and moxa tablets used in the prescription (i.e., it cannot perform chiral separation of dextrorotatory borneol and borneol), and does not include musk ketone, the main component of the main drug musk, in the content determination control index, which urgently needs to be improved.

[0020] Compared with the prior art, the advantage of the present invention is that a method based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) is used to detect the non-volatile components in the Xingnaojing injection, which can effectively detect the effective chemical components in the Xingnaojing injection.

[0021] The chromatographic conditions of the present invention are obtained through screening, and the screening method is as follows:

[0022] The mobile phase screening conditions are as follows:

[0023] A B 0.1-0.2% formic acid Acetonitrile 0.1-0.2% acetic acid Acetonitrile 0.1-0.2% formic acid Methanol 0.1-0.2% acetic acid Methanol

[0024] The mobile phase flow rate screening conditions were 0.1-0.3 mL / min, and the column temperature screening conditions were 25-35°C.

[0025] The mobile phase gradient screening conditions were as follows:

[0026] time B(v / v) B(v / v) B(v / v) B(v / v) 0-5min 5％ 6％ 5％ 4％ 5-45min 5％-30％ 6％-35％ 5％-35％ 4％-28％ 45-46min 30％-60％ 35％-70％ 35％-70％ 28％-55％ 46-50min 60％ 70％ 70％ 55％ 50-52min 60％-100％ 70％-100％ 70％-100％ 55％-100％ 52-60min 100％ 100％ 100％ 100％ 60-60.1min 5％ 6％ 5％ 4％ BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Base peak ion chromatogram (positive ion BPC graph) of chemical components in Xingnaojing injection detected by UHPLC-Q-Exactive Orbitrap MS

[0028] Figure 2 Curcuma dione (t R =50.94min) (top) and Xingnaojing injection (bottom) extracted ion current (positive ion EIC)

[0029] Figure 3 In t R =50.9min, the quasi-molecular ion m / z 237.18 ([C 15 H25 O2] + )

[0030] Figure 4 Germacrone (t R =54.16min) (top) and Xingnaojing injection (bottom) extracted ion current (positive ion EIC)

[0031] Figure 5 In t R =54.16min, the quasi-molecular ions of gemacic acid (upper) and Xingnaojing injection (lower) were m / z 219.18 ([C 15 H 23 O] + )

[0032] Figure 6 Curcumenol (t R =49.92min) (top) and Xingnaojing injection (bottom) extracted ion chromatogram (positive ion EIC diagram)

[0033] Figure 7 In t R =49.9min, the quasi-molecular ion m / z 235.17 ([C 15 H 23 O2] + )

[0034] Figure 8 Curcumol (t R =52.51min) (top) and Xingnaojing injection (bottom) extracted ion current (positive ion EIC)

[0035] Fig. 9 In t R =52.5min, the quasi-molecular ion m / z 237.18 ([C 15 H 25 O2] + )'s secondary mass spectrum. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below with reference to the embodiments.

[0037] Example 1

[0038] Weigh about 1 mg each of curcumenol, germacrone, curcumadione and curcumol, add appropriate amount of methanol to dissolve, and obtain a 0.1 mg / mL standard solution. The detection conditions are as follows.

[0039] Liquid phase conditions:

[0040] The chromatographic column was Waters BEH C18 (100×2.1 mm, 1.7 μm).

[0041] The mobile phase flow rate was set at 0.2 mL / min and the column temperature was set at 30 °C for separation of biological samples.

[0042] The mobile phase consisted of (A) 0.1% formic acid solution and (B) acetonitrile.

[0043] The mobile phase gradient was: 5% (v / v) B from 0 to 5 min, 5% to 30% B from 5 to 45 min, 30% to 60% B from 45 to 46 min, 60% to 60% B from 46 to 50 min, 60% to 100% B from 50 to 52 min, maintained at 100% B for 8 min, reduced to 5% B from 60 to 60.1 min, and the gradient elution re-equilibrium time was 10 min.

[0044] The injection volume was 15 μL.

[0045] Mass spectrometry conditions:

[0046] Scan mode: Full MS / dd-MS 2 .

[0047] In Full MS mode, the resolution is 140000; the AGC target is 1×10 6 , Maximum IT is 100 ms, and the scanning range is m / z 53.4-800.

[0048] The dd-MS2 / dd-SIM parameters are set to: resolution 17500, AGC target 1×10 5 , Maximum IT is 50ms, loop count is 10, isolation window is m / z 2; CE is 15, 25, 35eV. The ionization mode is ESI positive ion scanning, the ion source is HESI source, the ion source spray voltage is 3.0kV positive ion, the capillary temperature is 320℃, the sheath gas flow rate is 40 units N2, the auxiliary gas flow rate is 10 units N2, and the S-Lens RF level is 55.

[0049] Experimental Results

[0050] The base peak ion flow diagram (BPC diagram) of the chemical components of Xingnaojing injection shows that the content of chemical components in Xingnaojing injection is generally low. In its chromatogram, except for the chromatographic peaks of 6 components between 49min and 55min, no chromatographic peaks of other components in Xingnaojing injection were found. Although no obvious chromatographic peaks can be seen (the content of the compound is very low), by screening the LC-MS data of Xingnaojing injection at each time point one by one and based on the mass spectrometry fragmentation information, it can be inferred that there are more than 6 components in it. Finally, we found at least 40 chemical components in total. However, due to the unclear mass spectrometry fragmentation characteristics of these compounds and the large number of isomers, it is difficult to accurately judge their structures using only LC-MS. Therefore, only 4 reference substances (curcuma dione, germacron, curcumenol, and curcumol) were used to identify the chromatographic peaks of the 6 components between 49min and 55min. By comparing the extracted ion chromatogram (EIC) of the reference substance and Xingnaojing injection, comparing the retention time and predicted molecular formula, the fragment ions of the secondary mass spectrometry and their relative abundance, we identified four component peaks, which were determined to be four chemical components: curcumazone, germacron, curcumenol, and curcumol. Among them, curcumenol and curcumol were two chemical components that were not detected in the GC-MS test. The peak areas of these six component peaks accounted for the vast majority of the 40 components (at least more than 99%), and the proportion of the four component peaks identified by the reference substance, after calculating the peak area, accounted for about 66% of the total peak area of ​​the six component peaks. The extracted ion chromatogram (EIC) and mass spectra of the reference substances of curcumazone, germacron, curcumazone and curcumol are shown in the attached figure.

Claims

1. A method for detecting non-volatile chemical components in Xingnaojing injection, using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap mass spectrometry, characterized in that ,The method, the steps are as follows: Step 1, weighing curcuminol, germacrone, curcumadione and curcumol, adding appropriate amount of methanol to dissolve, and obtaining a standard solution, Step 2: Inject the standard solution and Xingnaojing injection into the chromatograph to obtain a chromatogram and perform LC-MS analysis as follows: Liquid phase conditions: The chromatographic column was Waters BEH C18, model 100 × 2.1 mm, 1.7 μm; The mobile phase flow rate was set to 0.2 mL / min and the column temperature was set to 30 °C for separation of biological samples; Mobile phase A was 0.1% formic acid solution, and mobile phase B was acetonitrile; The mobile phase gradient was: 5% B from 0 to 5 min, 5% to 30% B from 5 to 45 min, 30% to 60% B from 45 to 46 min, 60% B from 46 to 50 min, 60% to 100% B from 50 to 52 min, maintained at 100% B for 8 min, and decreased to 5% B from 60 to 60.1 min. The gradient elution re-equilibrium time was 10 min. The injection volume was 15 μL; Mass spectrometry conditions: Scan mode: Full MS / dd-MS 2 ; In Full MS mode, the resolution is 140000; the AGC target is 1×10 6 , Maximum IT is 100ms, and the scanning range is m / z 53.4-800; The dd-MS2 / dd-SIM parameters are set to: resolution 17500, AGC target 1×10 5 , Maximum IT is 50ms, loop count is 10, isolation window is m / z 2; CE is 15, 25, 35eV; The ionization mode was ESI positive ion scanning, the ion source was HESI source, the ion source spray voltage was 3.0 kV positive ion, the capillary temperature was 320°C, the sheath gas flow rate was 40 units N2, the auxiliary gas flow rate was 10 units N2, and the S-Lens RF level was 55.

Images