CN105203687A - Paeonol quick detection method - Google Patents

Abstract

The invention provides a rapid detection method of paeonol: 1) sample preparation; 2) PY-GC/MS detection; 3) data analysis and qualitative and quantitative analysis. The present invention selects PY-GC/MS technology to detect the content of paeonol, which is aimed at Chinese medicinal materials with paeonol as the main active ingredient, such as Cortex Moutan and Xu Changqing in actual production and life. To solve the problem of different quality, provide a rapid and trace detection method of paeonol, and then preliminarily judge the quality of such Chinese medicinal materials. The present invention has the characteristics of simple operation, low detection limit, good experimental reproducibility, intuitive and reliable experimental data, etc. , especially suitable for the detection of trace samples, and can be used for preliminary comparison of the quality differences between samples of different origins, varieties, batches of Moutan bark, Xu Changqing, etc., and has wide applicability.

Description

A kind of rapid detection method of paeonol

technical field

The invention relates to a method for rapidly detecting paeonol content based on fast pyrolysis-gas chromatography-mass spectrometry (PY-GC/MS method).

Background technique

Paeonol, also known as paeonol, is the main active ingredient in the root bark of Moutan of Ranunculaceae and the dry root or whole plant of Xu Changqing of Radixaceae. It has pharmacological effects such as sedative, hypnotic, antipyretic, analgesic, anti-inflammatory and antibacterial.

Recent studies have shown that paeonol has good preventive and therapeutic effects on cardiovascular and cerebrovascular diseases. Li Wei et al compared the effect of paeonol and aspirin on changing the hemorheology of rats, showing that paeonol can reduce the apparent viscosity of whole blood, hematocrit, red blood cell aggregation and platelet adhesion, and make the red blood cells The ability to deform was significantly enhanced; while the ability of aspirin to change the rheology of blood was more limited. This shows that paeonol can intervene in all aspects of thrombus formation. In addition, paeonol can inhibit the formation of strong carcinogens in a dose-dependent manner, especially in the process of carcinogenicity of aromatic amine compounds, and has played a role in inhibiting tumor growth. Paeonol and its derivatives are expected to be developed into a new generation of anti-tumor agents. drug. Therefore, rapid and efficient detection of paeonol in traditional Chinese medicines or their compound preparations is very important for drugability and modern application of traditional Chinese medicines.

The existing analysis and detection of paeonol mainly adopts the method of high performance liquid phase. Due to the limitations of analytical instruments, it is often necessary to carry out more complicated pretreatment on the samples. Paeonol is separated from medicinal materials or their compound preparations by extraction and other methods, and then analyzed by liquid chromatography methods such as HPLC/UPLC/SFCLC. The sample processing process of this analytical method is complex and the overall analysis time is long.

Rapid pyrolysis-gas chromatography/mass spectrometry (PY-GC/MS) is an efficient technique for detecting volatile components, which has developed rapidly in recent years. The pyrolysis process at low temperature is a very effective means of thermal separation. By controlling the temperature and time of pyrolysis, the preliminary gas-liquid or gas-solid phase separation of complex mixtures with low boiling points can be achieved; On-line and real-time sample preparation and primary separation capabilities. The pyrolysis time, temperature, heating rate, carrier gas flow rate, split ratio, ionization temperature, etc. during the analysis and detection process have a great influence on the analysis results. Therefore, based on the characteristics of PY-GC/MS technology, it is urgent to propose a rapid and trace detection method for paeonol.

Contents of the invention

The purpose of the present invention is to provide a rapid detection method of paeonol, and based on this, preliminarily judge the quality of Chinese medicinal materials such as Cortex Moutan and Xu Changqing and their preparations.

To achieve the above object, the present invention adopts the following technical solutions:

1) Accurately weigh 0.02-5.00 mg of Moutan Cortex, Xu Changqing or its compound preparation, and add the sample into the pyrolysis cup;

2) adopt fast pyrolysis-gas chromatography-mass spectrometry to measure the sample added in the pyrolysis cup;

3) After step 2), according to the measured original chromatographic data and mass spectrometry data, the chromatographic peak of paeonol in the sample is qualitatively determined, and the relative content of paeonol in the sample is obtained.

The sample is block or powder solid.

The pyrolysis conditions are as follows: the pyrolysis temperature is 180-300° C., and the pyrolysis time is 0.1-2 minutes.

The working conditions of the gas chromatography are as follows: the carrier gas is helium with a flow rate of 0.1-4.0mL/min, the split ratio is 0-500:1, and the heating program is: the initial temperature is 40-70°C, and it is kept for 0-3min, Then rise to 300°C at ≤20°C/min.

The working conditions of the mass spectrometer are as follows: EI source, positive ion detection, electron energy of 50-85eV, ion source temperature of 215-245°C, single quadrupole temperature of 145-185°C, full scan mode, The electron multiplier voltage is 1075-2980V, and the solvent delay is 0-6min.

The relative content is calculated using the area normalization method.

The qualitative of the chromatographic peak of described paeonol adopts NIST08 software automatic matching calculation.

The beneficial effects of the present invention are reflected in:

1) Rapid separation. The existing method is to preliminarily separate paeonol solution from Cortex Moutan or Xu Changqing or its preparation by extraction method, and after filtration, use liquid chromatography analysis, or thin-layer chromatography-spectroscopy technique (TLC-SP) to detect Its active ingredient; the general analysis time of this method is 4 to 24 hours. In the present invention, the low-boiling point substances of Cortex Moutan or Xu Changqing or its preparations are directly separated by thermal separation method, and paeonol is further separated by gas chromatography, which greatly saves the analysis time, and the conventional analysis time is 0.1 to 0.3 hours, improving analysis efficiency.

2) The sensitivity is good and the amount of sample required is small. The HPLC detection method is limited by the instrument, and generally needs to be separated first to obtain the crude product solution, so the required sample size is relatively large, usually ranging from several grams to tens of grams; analytical sensitivity.

3) The present invention optimizes the detection conditions of pyrolysis, chromatography, mass spectrometry, etc., can quickly characterize the required chromatographic peaks, and realizes rapid and accurate detection.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Aiming at the relatively low melting and boiling point of paeonol and its volatile characteristics, the present invention adopts rapid pyrolysis-gas chromatography/mass spectrometry (PY-GC/MS), fully combines thermal separation and chromatographic separation, and realizes paeonol Rapid separation of phenol, followed by qualitative and quantitative detection by mass spectrometry, to achieve the purpose of rapid and trace detection.

Example

A method for detecting paeonol based on PY-GC/MS, taking the Chinese medicinal material Cortex Moutan as an example, but the protection scope of the present invention is not limited to this embodiment, and the specific steps are as follows:

1. Sample preparation

Accurately weigh 1.00 mg (±0.02 mg) of the obtained peony bark (Shaanxi Yuwangtang Biotechnology Development Co., Ltd., Xi'an) into a 50 μL pyrolysis cup;

2. Pyrolysis:

The pyrolysis furnace adopts Frontiers PY2020is;

①Pyrolysis temperature: 200℃;

②Pyrolysis time: 0.20min.

3. Separation and detection

Chromatography:

Gas chromatography using Agilent 7890A:

①The analytical column is HP-5MS elastic quartz capillary column (30m*0.25id*0.25μm);

②Carrier gas: high-purity helium, the flow rate is 1.0mL/min;

③The sampling method is split injection, and the split ratio is 150:1;

④The temperature of the injection port is 280°C, and the temperature of the transfer line is 290°C;

⑤Procedural heating: the initial temperature is 60°C, keep it for 1min, and then increase to 300°C at 20°C/min.

Mass spectrometry (using Agilent 5975C):

①EI source, positive ion detection, electron energy is 70eV;

② The ion source temperature is 230°C, and the single quadrupole temperature is 150°C;

③The scanning mode is full scanning mode;

④ Electron multiplier voltage is 2042V, solvent delay is 3min.

4. Data analysis and processing

The retention time and integral area of the chromatogram are automatically completed by the workstation, and the chromatographic peaks are qualified using the NIST08 spectral library. The relative content of each volatile component was obtained by the area normalization method.

There are 23 identifiable peaks in the pyrolysis components of Cortex Moutan in this sample, and the components and their retention times are listed in Table 1. Retention time and peak area were calculated by Agilent Chemstation, and relative content was calculated by area normalization method.

Table 1. Composition and content of volatile components in Cortex Moutan (t _r =6.999: paeonol)

5. Precision, reproducibility and stability experiments

1. Precision experiment: with 5 parts of samples taken from the same Moutan bark, according to the above method, the samples were continuously injected 5 times, and 23 chromatographic peaks with a relative peak area greater than 0.5% were analyzed, and the relative retention time and relative peak area were analyzed. The RSDs (relative standard deviations) are respectively between 1.25-2.35% and 1.53-4.33%, indicating that the method of the present invention has good precision.

2. Reproducibility experiment: Take 5 samples of the same batch of Moutan cortex, and inject 5 times continuously according to the above method. Results The RSDs of the relative retention time and relative peak area of the 23 chromatographic peaks were between 0.56-4.37% and 1.48-3.99%, respectively, indicating that the method of the present invention has good reproducibility.

3. Stability test: take 1 prepared sample, inject and measure at 0, 2, 4, 8, 12, and 24 hours respectively. As a result, the relative retention time and relative peak area RSD of 23 chromatographic peaks are all less than 3.36% , illustrating that the method of the present invention is stable in 24h.

Due to the different places of origin, seasons, and processing methods of Chinese herbal medicines, their water content and density are slightly different, and the content of their compound preparations is also different. Therefore, the required pyrolysis, separation and analysis conditions are all different, and the condition range in the present invention is obtained accordingly.

The invention has the characteristics of simple operation, low detection limit, good experimental reproducibility, intuitive and reliable experimental data, etc., and is especially suitable for the detection of trace samples (sample amount can be as low as 20 μg), and can be used for the detection of paeonol-containing Chinese medicinal materials or their Quantitative comparison of the quality difference between preparation samples has wide applicability, and provides a reference idea and method for the quality standard and modern application of this kind of Chinese medicinal materials.

Claims (7)

1. a rapid detection method of paeonol, characterized in that: comprise the following steps: 1) Accurately weigh 0.02-5.00 mg of Moutan Cortex, Xu Changqing or its compound preparation, and add the sample into the pyrolysis cup; 2) adopt fast pyrolysis-gas chromatography-mass spectrometry to measure the sample added in the pyrolysis cup; 3) After step 2), according to the measured original chromatographic data and mass spectrometry data, the chromatographic peak of paeonol in the sample is qualitatively determined, and the relative content of paeonol in the sample is obtained. 2. the rapid detection method of a kind of paeonol according to claim 1, is characterized in that: described sample is bulk or powdery solid. 3. A rapid detection method for paeonol according to claim 1, characterized in that: the pyrolysis conditions are as follows: the pyrolysis temperature is 180-300°C, and the pyrolysis time is 0.1-2min. 4. the rapid detection method of a kind of paeonol according to claim 1, is characterized in that: the working condition of described gas chromatography is: carrier gas is the helium of flow velocity 0.1～4.0mL/min, and split ratio is 0～ 500:1, the heating program is as follows: the initial temperature is 40-70°C, keep it for 0-3 minutes, and then rise to 300°C at a rate of ≤20°C/min. 5. The rapid detection method of a kind of paeonol according to claim 1, is characterized in that: the working condition of described mass spectrum is: adopt EI source, positive ion detection, electron energy is 50～85eV, and ion source temperature is 215 ~245°C, the single quadrupole temperature is 145~185°C, the scan mode is full scan mode, the electron multiplier voltage is 1075~2980V, and the solvent delay is 0~6min. 6. the rapid detection method of a kind of paeonol according to claim 1, is characterized in that: described relative content adopts area normalization method to calculate. 7. the rapid detection method of a kind of paeonol according to claim 1, is characterized in that: the qualitative of the chromatographic peak of described paeonol adopts NIST08 software automatic matching calculation.