WO2022041796A1

WO2022041796A1 - Method for on-site identification of highly volatile chinese medicinal materials using surface acoustic wave/gas chromatography

Info

Publication number: WO2022041796A1
Application number: PCT/CN2021/088168
Authority: WO
Inventors: 赵军宁; 何世堂; 王剑波; 陆艳艳; 朱宏伟; 孙林; 刘久玲
Original assignee: 四川省中医药科学院; 中国科学院声学研究所
Priority date: 2020-08-31
Filing date: 2021-04-19
Publication date: 2022-03-03
Also published as: CN112180004A

Abstract

Provided is a method for on-site identification of highly volatile Chinese medicinal materials using surface acoustic wave/gas chromatography, comprising: (1) determining a sample volatility under pre-treatment conditions of ambient-temperature saturation treatment; (2) by means of a suitable method, pre-treating a sample; (3) obtaining a standard fingerprint spectrum; (4) testing a sample, to obtain a sample profile of the volatile components in a Chinese medicine sample to be tested, and comparing the sample profile to the standard fingerprint spectrum to identify the authenticity of the Chinese medicine to be tested. The invention has advantages such as simple sample pre-treatment, fast analysis, and on-site testing.

Description

A method for on-site identification of highly volatile Chinese medicinal materials using surface acoustic wave gas chromatograph

technical field

The present invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a method for on-site identification of highly volatile traditional Chinese medicinal materials using a surface acoustic wave gas chromatograph.

Background technique

Traditional Chinese medicine can be used for the prevention and treatment of various diseases. Because the quality of traditional Chinese medicine is affected by the origin, climate, environment, as well as processing, transportation, and storage, the quality of traditional Chinese medicine from different sources is uneven, especially for rare and precious traditional Chinese medicine. In order to obtain illegal benefits, criminals are increasingly adulterating and counterfeiting, which makes it difficult to distinguish the authenticity of traditional Chinese medicine from fake, the quality is unstable, and it is difficult to ensure the safety and effectiveness of the drug. Misuse of counterfeit and inferior products can lead to ineffectiveness in mild cases, and side effects in severe cases, even endangering people's lives, which greatly damages the quality and safety of traditional Chinese medicine and threatens people's health. At the same time, it also seriously damaged the image of traditional Chinese medicine and hindered the healthy growth of my country's traditional Chinese medicine industry. Therefore, it is an urgent need in the modern development stage of traditional Chinese medicine to actively carry out the authenticity identification and quality evaluation of traditional Chinese medicine, and develop various fast, reliable and easy-to-promote traditional Chinese medicine authenticity detection technologies and equipment.

At present, traditional Chinese medicine detection methods include character identification, microscopic identification, physical and chemical identification, and instrumental analysis methods such as gas chromatography, high performance liquid chromatography, and near-infrared spectroscopy. Although intuitive empirical identification and microscopic identification are simple and fast, it is necessary to identify Those who have considerable experience and equipment such as microscopes are only suitable for traditional Chinese medicines and powdered traditional Chinese medicines with obvious characteristics in appearance. Extraction, separation, concentration, drying, etc., the test takes a long time (30-60min), requires high equipment and equipment, and needs to be carried out in the laboratory, which is difficult to popularize and promote, and cannot achieve the purpose of rapid inspection. Due to the variety and complex sources of traditional Chinese medicines, and the large sample size for market testing, rapid on-site testing can effectively shorten the testing time and reduce labor costs. At present, the existing on-site rapid inspection methods of traditional Chinese medicine include appearance identification, character identification, chemical identification and thin layer identification. However, its sensitivity is poor, its anti-interference ability is weak, and it is prone to false positive or false negative results. Therefore, it is of great practical significance to establish a fast and accurate on-site detection method of traditional Chinese medicine.

SUMMARY OF THE INVENTION

Aiming at the problem of the lack of fast and accurate on-site detection methods for traditional Chinese medicines in the prior art, the present invention provides a method for on-site identification of high-volatile traditional Chinese medicinal materials by using a surface acoustic wave gas chromatograph, the purpose of which is to provide a method that can be separated from laboratory conditions, On-site (such as the origin of traditional Chinese medicine, the market of traditional Chinese medicinal materials, etc.) to identify the authenticity of traditional Chinese medicine. In this method, after a simple determination of the volatility of the components in Chinese medicinal materials, the Chinese medicinal materials are subjected to simple pretreatment, retention index calibration and headspace sampling, combined with surface acoustic wave fast gas chromatograph (GC-SAW), The rapid analysis and high sensitivity characteristics of surface acoustic wave fast gas chromatography are used to realize the analysis of the characteristic components of traditional Chinese medicine. The method has the advantages of simple sample pretreatment, fast analysis speed, and on-site detection, and can be applied to on-site authenticity identification of traditional Chinese medicines.

A method for on-site identification of Chinese medicinal materials using a surface acoustic wave gas chromatograph, comprising the following steps:

(1) Determination of sample volatility: take a sample of traditional Chinese medicine to be tested and carry out saturation treatment at room temperature to obtain a sample to be determined; use a surface acoustic wave gas chromatograph to detect the sample to be determined to obtain a spectrum of volatile components in the sample to be determined. ; According to the response value of the largest peak of the response value, the TCM sample to be tested is judged as a high volatility sample, a medium volatility sample or a low volatility sample;

(2) Sample pretreatment: according to the judgment result, the sample of the traditional Chinese medicine to be tested and its corresponding standard product are subjected to pretreatment to obtain the sample to be tested and the standard sample, and the pretreatment condition of the high volatility sample is normal temperature saturation treatment; The pretreatment condition of the volatile sample is high temperature saturation treatment; the pretreatment condition of the low volatility sample is solid phase extraction;

(3) Obtain the standard fingerprint: use the surface acoustic wave gas chromatograph to detect the standard sample to obtain the standard fingerprint of the volatile components in the standard;

(4) Detection of the sample: use a surface acoustic wave gas chromatograph to detect the sample to be tested, obtain a sample spectrum of volatile components in the traditional Chinese medicine sample to be tested, and compare the sample spectrum with the standard fingerprint spectrum obtained in step (3), To identify the authenticity of the Chinese medicine to be tested.

Preferably, before step (1), a surface acoustic wave gas chromatograph is also used to detect the normal alkane mixed standard, and record the peak time of each normal alkane standard for use in step (1) , step (3) or step (4) obtain the retention index of each volatile component in the process of obtaining collection of illustrative plates;

The retention index is calculated as follows:

Among them, RI is the retention index of volatile components, t _x is the retention time of volatile components, and n and n+1 are the carbon atoms of two standard n-alkanes whose peaks are adjacent to the peaks of volatile components, respectively. number, t _n and t _n+1 are the retention times of the peaks of the two standard products of n-alkanes adjacent to the spectral peaks of the volatile components, respectively.

Preferably, in step (1), the standard for judging the volatility of the sample is: the response value of the maximum response peak in the spectrum is greater than or equal to 2000Hz, which is a high volatility sample; the response value of the maximum response peak in the spectrum is less than 2000Hz, and greater than or equal to 2000Hz or equal to 100Hz is a medium volatility sample; the response value of the maximum response peak in the spectrum is less than 100Hz is a low volatility sample.

Preferably, in step (1) or step (2), the specific process of the normal temperature saturation treatment is as follows: take 20-1000 mg of traditional Chinese medicine samples and put them in a 10-40 ml headspace bottle, and saturate at room temperature for 1-60 min; in the step (2) , the specific process of high temperature saturation treatment is, take 20-1000mg traditional Chinese medicine sample and put it into 10-40ml headspace bottle, and saturate it at a temperature of 30-100 ℃ for 1-60min; in the step (2), the specific process of solid phase extraction is, Take 20-1000mg Chinese medicine samples into a 10-40ml headspace vial, and use a solid-phase extraction needle to extract for 5s-5min.

Preferably, in step (1), step (3) or step (4), the test process of the surface acoustic wave gas chromatograph is: in the sample injection state, the sampling port is connected to the sample to be tested, and the sampling pump is started, and the The test sample is sucked into the pre-concentration tube; in the detection state, the six-way valve is switched to make the carrier gas flow through the pre-concentration tube, and carry the sample to be tested in the pre-concentration tube into the chromatographic column and the detector in turn to complete the detection.

Preferably, the surface acoustic wave gas chromatography column is selected from DB-5, SPB-5, Rtx-5, BP-5, OV-5, 007-2 (MPS-5), SE-52, SE-54, XTI- 5. PTE-5, ZB-5, AT-5, MDN-5 or ZB-5, the length of the chromatographic column is 1-10 meters.

Preferably, the detection conditions of the surface acoustic wave gas chromatograph are as follows: the initial temperature of the chromatographic column is 40-50°C, and during the detection process, the chromatograph is subjected to a heating program of 0-20°C/s The temperature of the column was raised to 200°C; the flow rate of the column was 1-7 mL/min, and the carrier gas was nitrogen or helium.

Preferably, in the sampling state of the surface acoustic wave gas chromatograph, the working time of the sampling pump is 5-60s.

Preferably, the detection conditions of the surface acoustic wave gas chromatograph are as follows: the temperature of the detector is 25-60°C.

Preferably, in step (4), using similarity evaluation or principal component analysis method, the sample spectrum is compared with the standard fingerprint spectrum obtained in step (3) to identify the authenticity of the Chinese medicine to be tested.

Preferably, for highly volatile samples, a method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph, comprising the following steps:

(1) Determination of sample volatility: take a sample of traditional Chinese medicine to be tested and carry out saturation treatment at room temperature to obtain a sample to be determined; use a surface acoustic wave gas chromatograph to detect the sample to be determined to obtain a spectrum of volatile components in the sample to be determined. ; According to the size of the response value of the largest peak of the response value, determine whether the sample to be tested is a highly volatile sample; the standard for determining the volatility of the sample is: the response value of the largest peak of the response value in the spectrum is greater than or equal to 2000 Hz is a highly volatile sample;

(2) Sample pretreatment: after determining that the sample to be tested is a highly volatile sample, the sample of the traditional Chinese medicine to be tested and its corresponding standard are pretreated to obtain the sample to be tested and the standard sample, and the pretreatment condition is normal temperature saturation treatment;

(4) Detection of the sample: use a surface acoustic wave gas chromatograph to detect the sample to be tested, obtain a sample spectrum of volatile components in the traditional Chinese medicine sample to be tested, and compare the sample spectrum with the standard fingerprint spectrum obtained in step (3), To identify the authenticity of the Chinese medicine to be tested. The traditional Chinese medicine samples, the traditional Chinese medicine samples to be tested and the standard products mentioned in the present invention refer to the traditional Chinese medicinal materials that have been pre-cut, pulverized, sheared or trimmed to a suitable size. The samples to be tested, the samples to be tested and the standard samples refer to the gaseous or solid phase extraction samples that can be directly used for testing by a surface acoustic wave gas chromatograph after the Chinese medicinal materials are processed by the pretreatment method provided by the present invention. The "normal temperature" in the present invention refers to a condition in which additional heating or cooling is not performed under the air temperature at the detection site.

Compared with traditional laboratory testing, the difficulty of on-site testing is that due to the influence of factors such as geographical environment, climatic conditions and prevailing weather conditions, the uncertainty of the most suitable pretreatment and testing conditions for specific Chinese herbal medicines has increased. The state of the equipment changes greatly, which affects the accuracy of the detection. Based on the above difficulties, the present invention firstly determines the volatility of the sample before testing, and determines the appropriate pre-treatment method for medicinal materials under the local geographical, climatic and weather conditions according to certain standards, so as to ensure that each sample in the sample to be tested is The content of volatile components. Further, through the comparison with the standard sample, the fluctuation of the instrument state can be overcome, and the detection accuracy can be improved. Through the calibration of n-alkane mixed standard sample, the peak time of volatile components can be converted into retention index, which makes each spectral peak more intuitive and standardized, which facilitates the acquisition of standard fingerprints and facilitates on-site qualitative judgment.

Therefore, the method provided by the embodiment of the present invention has at least the following advantages:

1. Be able to choose the most effective pretreatment conditions and detection conditions to ensure the accuracy of detection.

2. The sample pretreatment process is simple, and the portable surface acoustic wave gas chromatograph used has a fast analysis speed. It only takes 2-8 minutes for a complete test of a sample, which is suitable for rapid on-site detection and screening work;

3. The test speed is fast, the operation is simple, no organic reagents are required, it is environmentally friendly, and there is no secondary pollution;

4. The operating cost of the instrument is very low, and there are few consumer goods, which is suitable for large-scale on-site testing work;

5. For medium-volatile samples, the high-temperature saturation method has greatly improved the detection sensitivity of the portable surface acoustic wave gas chromatograph, and can complete the detection of traditional Chinese medicines with less volatile components. For low-volatile samples, the detection sensitivity of the portable surface acoustic wave gas chromatograph can be greatly improved by using solid-phase microextraction, and the detection of traditional Chinese medicines with few volatile components can be completed.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 shows the structural schematic diagram of the surface acoustic wave gas chromatograph in the sample injection state;

Fig. 2 shows the structural schematic diagram of the surface acoustic wave gas chromatograph in the detection state;

Fig. 3 has shown embodiment 1 Chinese medicine musk detection result spectrum;

Figure 4 shows the chromatogram of musk standard 1 in Example 1 and the retention index and response value of each peak;

Fig. 5 has shown embodiment 2 Chinese medicine turmeric detection result spectrum;

Fig. 6 has shown the chromatogram of Sichuan Ji An Tang turmeric and the retention index and response value of each peak in embodiment 2;

Fig. 7 has shown embodiment 3 Chinese medicine Cordyceps sinensis detection result spectrum;

Fig. 8 has shown the chromatogram of Anhui Cordyceps in Example 3 and the retention index and response value of each peak;

Fig. 9 has shown the detection result spectrum of traditional Chinese medicine Panax notoginseng in Example 4;

Figure 10 shows the chromatogram of 8-3 Panax notoginseng in Example 4 and the retention index and response value of each peak

Fig. 11 has shown embodiment 5 Chinese medicine Bezoar detection result spectrum;

Figure 12 shows the chromatogram of 5-1 Bezoar and the retention index and response value of each peak in Example 5;

Figure 13 has shown the Chinese medicine bergamot detection result spectrum of embodiment 6;

Figure 14 shows the chromatogram of bergamot 10-2 in Example 6 and the retention index and response value of each peak.

detailed description

The surface acoustic wave gas chromatograph used in the embodiment of the present invention is a portable surface acoustic wave gas chromatograph. As shown in Figures 1 and 2, the surface acoustic wave gas chromatograph includes a sampling port 1, a sampling pump 2, a pre-concentration tube 3, a six-way valve 4, a carrier gas bottle 5, a chromatographic column 6, a detector 7 and a data processing device 8.

The SAW gas chromatograph can be in the injection state or the detection state, and can switch between these two states.

As shown in FIG. 1 , when the SAW gas chromatograph is in the sampling state, the first air port 41 of the six-way valve 4 is connected to the first air port 31 of the pre-concentration pipe 3 , and the second air port 42 of the six-way valve 4 is connected to The carrier gas bottle 5, the third air port 43 of the six-way valve 4 is connected to the air inlet of the chromatographic column 6, the fourth air port 44 of the six-way valve 4 is connected to the second air port 32 of the pre-concentration pipe 3, and the fifth air port of the six-way valve 4 is connected. The air port 45 is connected to the sampling pump 2 , and the sixth air port 46 of the six-way valve 4 is connected to the sampling port 1 .

Through the air pressure difference formed by the sampling pump 2, the sample enters the pre-concentration tube 3 through the sampling port 1 to complete the sampling.

After the injection is completed, rotate the six-way valve 4 to switch the SAW gas chromatograph to the injection state, as shown in Figure 2. In the sampling state, the first air port 41 of the six-way valve 4 is connected to the second air port 32 of the pre-concentration pipe 3, the second air port 42 of the six-way valve 4 is connected to the sampling pump 2, and the third air port 43 of the six-way valve 4 is connected to the sampling pump. Port 1, the fourth air port 44 of the six-way valve 4 is connected to the first air port 31 of the pre-concentration pipe 3, the fifth air port 45 of the six-way valve 4 is connected to the carrier gas bottle 5, and the sixth air port 46 of the six-way valve 4 is connected to the chromatographic column. 6 air intakes.

At this time, from the carrier gas cylinder 5, a gas path is formed that passes through the pre-concentration tube 3, the chromatography column 6, and the detector 7 in this order. During the detection process, the pre-concentration tube 3 is heated to vaporize the sample therein. The vaporized sample in the pre-concentration tube 3 can enter the chromatographic column 6 with the carrier gas to complete the gas phase separation, and then enter the detector 7 to complete the detection of each gas phase component after the sample separation. The data detected by the detector 7 can be sent to the data processing device 8, and the data processing device 8 processes and presents the data, so that the detection personnel can know the detection result.

During the detection process, the experimental conditions of the SAW gas chromatograph are as follows:

Use DB-5 chromatographic column; the temperature program of the chromatographic column is: the initial temperature is maintained at 40-50 °C, and the temperature is increased to 200 °C at 0-20 °C/s;

The flow rate of DB-5 chromatographic column is 1-7ml/min;

The length of DB-5 chromatographic column is 1-10 meters;

The inlet temperature is 120-200℃;

The temperature of the six-way valve 4 is 120-165°C;

The detector temperature is 25-60℃;

The pumping time of the sampling pump 2 is 5-60s;

The carrier gas bottle 5 is nitrogen or helium.

As a kind of preferred scheme, the experimental condition of the surface acoustic wave gas chromatograph that embodiment 1-6 adopts is as follows:

DB-5 chromatographic column was used; the temperature program of the chromatographic column was as follows: the initial temperature was maintained at 40°C, and the temperature was raised to 200°C at 10°C/s;

The flow rate of DB-5 chromatographic column is 4ml/min;

The size of the DB-5 chromatographic column is 1m×0.25mm×0.25μm;

The inlet temperature is 200°C;

The temperature of the six-way valve 4 is 160°C;

The detector temperature is 40°C;

The pumping time of the sampling pump 2 is 10s;

The carrier gas bottle 5 is nitrogen.

The technical solutions of the present invention will be further described below through specific embodiments.

Example 1

The present embodiment detects musk, including the following steps:

(1) Retention index calibration: In this embodiment, the retention index is used to formulate a digital fingerprint, which can effectively avoid errors caused by differences in operating factors and experimental conditions. Take 20 mg of 13 kinds of n-alkane mixed standard (C6-C18), add it into a 40ml headspace bottle, and after saturating it at room temperature for 5 minutes, connect the headspace bottle to a portable surface acoustic wave gas chromatograph, so that the gas sample can pass through the pump It is sucked into the chromatograph and separated on the chromatographic column. The detection is carried out, the detection signal is obtained on the surface acoustic wave detector, and the retention time of each normal alkane standard is recorded. The peak time of the n-alkane standard is used to calculate the retention index of the volatile components of the sample to be tested using Kovats' retention index RI calculation formula in the subsequent step. The formula for calculating the retention index RI of Kovats is shown in formula (1).

(2) Determination of sample volatility: In order to ensure that a signal with sufficient intensity is collected, different volatile Chinese medicines need to be pretreated in different ways. When the inspectors need to detect traditional Chinese medicines with unknown volatility, the following scheme can be used to determine the suitable pretreatment method of traditional Chinese medicines.

The standard samples of the traditional Chinese medicine samples to be tested were pretreated by the normal temperature saturation treatment method. The normal temperature saturation treatment method is specifically, take 20mg sample and put it into a 40ml headspace bottle, and obtain the sample to be tested after being saturated for 5min at normal temperature (detection site temperature, can be approximately considered to be 25°C in this embodiment). Use a surface acoustic wave gas chromatograph to detect the sample to be side, and observe the detection signal.

In this embodiment, the response value of the peak with the largest response value is greater than 2000 Hz, and it can be determined that the traditional Chinese medicine sample is a high-volatile traditional Chinese medicine sample, and the suitable pretreatment method is normal temperature saturation treatment.

(3) headspace bottle background detection: in the present embodiment, the process of using the portable surface acoustic wave gas chromatograph to detect the blank background of the headspace bottle is as follows: get 40ml headspace bottle, connect with the portable surface acoustic wave gas chromatograph, top The air in the empty bottle is sucked into the chromatograph by the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. The obtained blank background has no signal interference at the characteristic peaks of traditional Chinese medicine.

(4) standard product analysis experiment: the present embodiment adopts the musk purchased from different manufacturers as the standard product, which is respectively recorded as musk standard product 1, musk standard product 2, musk standard product 3, musk standard product 4 and musk standard product 5, Take 20 mg of each of the five standard products and add them to a 40 ml headspace bottle. After saturating for 5 minutes at room temperature, connect the headspace bottle to a portable surface acoustic wave gas chromatograph. separated, and a detection signal was obtained on a surface acoustic wave detector. After obtaining the detection signal, the data processing device can calculate the retention index of the active ingredient in the musk standard according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the musk standard. The data processing unit can also calculate the response (peak area) of the active ingredient in the musk standard. According to the sample peak retention index and response obtained in this example, a database of traditional Chinese medicine musk fingerprints was established. Among them, the response, that is, the peak area, can reflect the content of the corresponding active ingredient.

(5) Analysis experiment of samples of traditional Chinese medicine to be tested: In this example, the unknown musk sample was taken as the sample of traditional Chinese medicine to be tested, 20 mg of the sample of traditional Chinese medicine to be tested was taken, added to a 40 ml headspace bottle, and after being saturated at room temperature for 5 minutes, the headspace bottle was combined with a portable The surface acoustic wave gas chromatograph is connected, the gas to be tested is sucked into the chromatograph through the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. After obtaining the detection signal, the data processing device can calculate the retention index of the active ingredient in the detected musk according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the detected musk. The data processing unit can also calculate the response (peak area) of the active ingredient in the musk standard. According to the comparison between the obtained sample peak retention index and the traditional Chinese medicine musk fingerprint database established in step (4), the authenticity is identified by observing the similarity of the position and relative size of the spectral peaks of the main components.

Wherein, Figure 3 shows the chromatogram of each musk detected in step (5). Figure 4 shows the chromatogram of Musk Standard 1 along with the retention index and response values for each peak. From the comparison of each spectrum in the figure, it can be clearly identified that the TCM sample to be tested is genuine musk. The experimental results in this embodiment show that the method of the present invention can accurately detect and identify whether the Chinese medicine sample to be tested is genuine or counterfeit.

Example 2

The present embodiment detects turmeric, comprising the following steps:

Among them, RI is the retention index of volatile components, t _x is the retention time of volatile components, and n and n+1 are the carbon atoms of two standard n-alkanes whose peaks are adjacent to the peaks of volatile components, respectively. number, t _n and t _n+1 are the retention times of the peaks of the standard products of the two normal alkanes adjacent to the spectral peaks of the volatile components, respectively.

The standard samples of the traditional Chinese medicine samples to be tested were pretreated by the normal temperature saturation treatment method. The normal temperature saturation treatment method is as follows: take 450 mg of the sample and put it into a 40 ml headspace bottle, and obtain the sample to be tested after saturation for 5 minutes at normal temperature (the temperature at the detection site, which can be approximately considered to be 25° C. in this embodiment). Use a surface acoustic wave gas chromatograph to detect the sample to be side, and observe the detection signal.

(4) Standard product analysis experiment: take 450 mg of standard product Guangxi turmeric, standard product Anhui turmeric, standard product Sichuan Ji'antang turmeric and standard product Sichuan Tongrentang turmeric, add 40ml headspace bottle, and after saturating for 5 minutes at room temperature, the top The empty bottle is connected to the portable surface acoustic wave gas chromatograph, and the gas to be tested is sucked into the chromatograph through the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. After the detection signal is obtained, the data processing device can calculate the retention index of the active ingredient in the turmeric standard product according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the turmeric standard product. The data processing unit can also calculate the response (peak area) of the active ingredient in the turmeric standard. According to the sample peak retention index and response obtained in this example, a database of traditional Chinese medicine turmeric fingerprints was established. Among them, the response, that is, the peak area, can reflect the content of the corresponding active ingredient.

(5) Analysis experiment of samples of traditional Chinese medicines to be tested: In this example, the unknown samples were taken as samples of traditional Chinese medicines to be tested, 450 mg of the samples of traditional Chinese medicines to be tested were taken, put into a 40 ml headspace bottle, and after saturating for 5 minutes at room temperature, the headspace bottle and the portable acoustic The surface wave gas chromatograph is connected, the gas to be tested is sucked into the chromatograph through the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. After obtaining the detection signal, the data processing device can calculate the retention index of the active ingredient in the detected turmeric according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the detected turmeric. The data processing unit can also calculate the response (peak area) of the active ingredient in the turmeric standard. According to the comparison between the obtained sample peak retention index and the traditional Chinese medicine turmeric fingerprint database established in step (4), the authenticity is identified by observing the similarity of the position and relative size of the spectral peaks of the main components.

Wherein, Figure 5 shows the chromatogram of each turmeric detected in step (5). Figure 6 shows the chromatogram of the standard Sichuan Jiantang turmeric and the retention index and response value of each peak. From the comparison of each spectrum in the figure, it can be clearly identified that the TCM samples to be tested are genuine turmeric. The experimental results in this embodiment show that the method of the present invention can accurately detect and identify whether the Chinese medicine sample to be tested is genuine or counterfeit.

Example 3

The present embodiment detects Cordyceps sinensis, comprising the following steps:

(1) Retention index calibration: In this embodiment, the retention index is used to formulate a digital fingerprint, which can effectively avoid errors caused by differences in operating factors and experimental conditions. Take 20mg of 13 kinds of n-alkane mixed standard (C6-C18), put it into a 40ml headspace bottle, and saturate it in a constant temperature device at 60 °C for 5min. The air in the bottle is sucked into the chromatograph by the pump and separated on the chromatographic column. The detection is carried out, and a detection signal is obtained on a surface acoustic wave detector, thereby obtaining and recording the retention time of each normal paraffin standard. The peak time of the n-alkane standard is used to calculate the retention index of the volatile components of the sample to be tested using Kovats' retention index RI calculation formula in the subsequent step.

The formula for calculating the retention index RI of Kovats is shown in formula (1).

(2) The same method as in Example 1 was used to determine the volatility of the sample. In this embodiment, the response value of the peak with the largest response value is between 100 Hz and 2000 Hz. It can be determined that the Chinese medicine sample is a medium volatile Chinese medicine sample, and the suitable pretreatment method is high temperature saturation treatment.

(3) High-temperature background detection of headspace bottle: In the present embodiment, the process of using a portable surface acoustic wave gas chromatograph to detect a blank Chinese medicine sample is as follows: take a 40ml headspace bottle, saturate it in a 60°C constant temperature device for 5 minutes, and use a portable surface acoustic wave gas chromatograph The surface acoustic wave gas chromatograph is connected, and the air in the headspace bottle is sucked into the chromatograph through the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. The obtained blank background has no signal interference at the characteristic peaks of traditional Chinese medicine.

(4) Standard product analysis experiment: Take 20 mg of Anhui Cordyceps sinensis, Sichuan Cordyceps sinensis and Yulin Cordyceps sinensis standard products, add them into a 40ml headspace bottle, and saturate them in a constant temperature device at 60°C for 5 minutes, then connect the headspace bottle to a portable surface acoustic wave gas chromatograph. Connected, the gas to be tested is sucked into the chromatograph through the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. After obtaining the detection signal, the data processing device can calculate the retention index of the active ingredient in the Cordyceps sinensis standard product according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the Cordyceps sinensis standard product. The data processing device can also calculate the response (peak area) of the active ingredient in the Cordyceps sinensis standard. According to the sample peak retention index and response obtained in this example, a database of traditional Chinese medicine Cordyceps sinensis fingerprints was established. Among them, the response, that is, the peak area, can reflect the content of the corresponding active ingredient.

(5) Analysis experiment of Chinese medicine samples to be tested: In this example, false Cordyceps sinensis (counterfeit product) is taken as the Chinese medicine sample to be tested, 20 mg of false Cordyceps sinensis (counterfeit product) is taken, added to a 40ml headspace bottle, and saturated for 5min in a constant temperature device at 60°C Then, connect the headspace bottle to the portable surface acoustic wave gas chromatograph, and the gas to be tested is sucked into the chromatograph by the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. After obtaining the detection signal, the data processing device can calculate the retention index of the active ingredient in the detected Cordyceps sinensis according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the detected Cordyceps sinensis. The data processing device can also calculate the response (peak area) of the active ingredient in the Cordyceps sinensis standard. According to the comparison between the obtained sample peak retention index and the fingerprint database of Chinese medicine Cordyceps sinensis established in step (4), the authenticity is identified by observing the similarity of the position and relative size of the spectral peaks of the main components.

Wherein, Figure 7 shows the chromatogram of each Cordyceps sinensis detected in step (5). Figure 8 shows the chromatogram of Anhui Cordyceps sinensis and the retention index and response value of each peak. From the comparison of each spectrum in the figure, it can be clearly identified that the Chinese medicine sample to be tested is false Cordyceps sinensis. The experimental results in this embodiment show that the method of the present invention can accurately detect and identify whether the Chinese medicine sample to be tested is genuine or counterfeit.

In addition, in this embodiment, the use of high temperature saturation method for pretreatment of medium volatile samples has a great improvement in the sensitivity of SAW gas chromatography detection compared to the use of normal temperature saturation for sample pretreatment. It only takes 7 minutes.

Example 4

The present embodiment detects Panax notoginseng, including the following steps:

(2) The same method as in Example 1 was used to determine the volatility of the sample. In the present embodiment, the response value of the peak with the largest response value is between 100Hz-2000Hz, it can be determined that the Chinese medicine sample is a medium volatile Chinese medicine sample, and its suitable pretreatment method is high temperature saturation treatment.

(4) Standard product analysis experiment: buy Panax notoginseng flowers from three different manufacturers, numbered as 8-1 Panax notoginseng flower, 8-2 Panax notoginseng flower, 8-3 Panax notoginseng flower, the above three kinds of bezoar are used as standard products for each Take 20mg, add it into a 40ml headspace bottle, and saturate it in a constant temperature device at 60°C for 5 minutes. Connect the headspace bottle to a portable surface acoustic wave gas chromatograph. The gas to be tested is sucked into the chromatograph by a pump and separated on a chromatographic column. And the detection signal is obtained on the surface acoustic wave detector. After the detection signal is obtained, the data processing device can calculate the retention index of the active ingredient in the Panax notoginseng standard product according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the Panax notoginseng standard product. . The data processing device can also calculate the response (peak area) of the active ingredient in the Panax notoginseng flower standard. According to the sample peak retention index and response obtained in this example, a fingerprint database of traditional Chinese medicine Panax notoginseng was established. Among them, the response, that is, the peak area, can reflect the content of the corresponding active ingredient.

(5) Analysis experiment of samples of traditional Chinese medicines to be tested: In this example, the unknown authenticity of Panax notoginseng was used as the samples of traditional Chinese medicines to be tested, 20 mg of the samples of traditional Chinese medicines to be tested were taken, added to a 40ml headspace bottle, and saturated in a constant temperature device at 60°C for 5 min. The headspace bottle is connected to the portable surface acoustic wave gas chromatograph, the gas to be tested is sucked into the chromatograph through the pump, separated on the chromatographic column, and the detection signal is obtained on the surface acoustic wave detector. After obtaining the detection signal, the data processing device can calculate the retention index of the active ingredient in the detected Panax notoginseng according to the retention time of the normal alkane standard product calibrated in step (1) and the retention time of the active ingredient in the detected Panax notoginseng flower. . The data processing device can also calculate the response (peak area) of the active ingredient in the Panax notoginseng flower standard. According to the comparison between the obtained sample peak retention index and the fingerprint database of traditional Chinese medicine Panax notoginseng established in step (4), the authenticity is identified by observing the similarity of the position and relative size of the spectral peaks of the main components.

Wherein, Figure 9 shows the chromatogram of each Panax notoginseng detected in step (5). Figure 10 shows the chromatogram of Anhui Panax notoginseng and the retention index and response value of each peak. From the comparison of each spectrum in the figure, it can be clearly identified that the samples of traditional Chinese medicine to be tested are genuine Panax notoginseng flowers. The experimental results in this embodiment show that the method of the present invention can accurately detect and identify whether the Chinese medicine sample to be tested is genuine or counterfeit.

In addition, in this embodiment, the use of high-temperature saturation method for pretreatment of medium-volatile samples greatly improves the sensitivity of SAW gas chromatography detection compared to the use of normal temperature saturation for sample pretreatment, and the detection time of each traditional Chinese medicine sample is It only takes 7 minutes.

Example 5

The present embodiment detects Bezoar, comprising the following steps:

(1) Retention index calibration: In this embodiment, the retention index is used to formulate a digital fingerprint, which can effectively avoid errors caused by differences in operating factors and experimental conditions. Take 20 mg of 13 kinds of n-alkane mixed standard (C6-C18), put it into a 40 ml headspace sample bottle, use a solid-phase microextraction needle (in this example, use fused silica optical fiber) to extract for 20s, then the solid-phase microextraction needle It is connected with the port of the portable surface acoustic wave gas chromatograph, and the gas to be tested in the solid-phase microextraction needle is sucked into the chromatograph through the pump and separated on the chromatographic column. The detection is carried out, and the detection signal is obtained on the surface acoustic wave detector, thereby obtaining and recording the peak time of each n-alkane standard product. The retention time of the normal alkane standard product peak is used to calculate the retention index of the volatile components of the sample to be tested using Kovats' retention index RI calculation formula in the subsequent step. The formula for calculating the retention index RI of Kovats is shown in formula (1).

(2) The same method as in Example 1 was used to determine the volatility of the sample. In this embodiment, the response value of the peak with the largest response value is less than 100 Hz, and it can be determined that the traditional Chinese medicine sample is a low-volatile traditional Chinese medicine sample, and the suitable pretreatment method is solid phase extraction.

(3) Headspace bottle solid-phase extraction background detection: In the present embodiment, the process of using a portable surface acoustic wave gas chromatograph to detect a blank Chinese medicine sample is as follows: take a 40ml headspace bottle, use a solid-phase microextraction needle to extract for 20s, Use a solid-phase microextraction needle to connect with a portable surface acoustic wave gas chromatograph, suck it into the chromatograph through a pump, separate it on a chromatographic column, and obtain a detection signal on a surface acoustic wave detector. The obtained blank background has no signal interference at the characteristic peaks of traditional Chinese medicine.

(4) Standard product analysis experiment: Buy bezoar from five different manufacturers, numbered 5-1 bezoar, 5-2 bezoar, 5-3 bezoar, 5-4 bezoar and 5-5 bezoar, and the above five bezoars are used as the standard Take 20mg of each product, add it into a 40ml headspace bottle, use a solid-phase microextraction needle to extract for 20s, connect the solid-phase microextraction needle to a portable surface acoustic wave gas chromatograph, and the gas to be tested is sucked into the chromatograph through a pump. Separation is carried out on the column, and the detection signal is obtained on the surface acoustic wave detector. After the detection signal is obtained, the data processing device can calculate the retention index of the active ingredient in the standard bezoar according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the standard bezoar. The data processing device can also calculate the response (peak area) of the active ingredient in the Bezoar standard. According to the sample peak retention index and response obtained in this example, a database of traditional Chinese medicine bezoar fingerprints was established. Among them, the response, that is, the peak area, can reflect the content of the corresponding active ingredient.

(5) Analysis experiment of samples of traditional Chinese medicine to be tested: In this example, a fake bezoar (pseudo bezoar) was used as the sample of traditional Chinese medicine to be tested. Take 20 mg of fake bezoar (pseudo bezoar), add it into a 40ml headspace bottle, use a solid-phase microextraction device for extraction for 20s, and connect it to a portable surface acoustic wave gas chromatograph. separation, and obtain the detection signal on the surface acoustic wave detector. After the detection signal is obtained, the data processing device can calculate the retention index of the active ingredient in the detected bezoar according to the retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the detected bezoar. The data processing device can also calculate the response (peak area) of the active ingredient in the Bezoar standard. According to the comparison between the obtained sample peak retention index and the traditional Chinese medicine bezoar fingerprint database established in step (4), the authenticity is identified by observing the similarity of the position and relative size of the spectral peaks of the main components.

Among them, Figure 11 shows the chromatogram of each bezoar detected in step (5). Figure 12 shows the chromatogram of 5-1 Bezoar and the retention index and response value of each peak. From the comparison of each spectrum in the figure, it can be clearly identified that the Chinese medicine sample to be tested is false bezoar. The experimental results in this embodiment show that the method of the present invention can accurately detect and identify whether the Chinese medicine sample to be tested is genuine or counterfeit.

In addition, in this embodiment, the use of solid-phase microextraction device for pretreatment of low-volatile samples has a great improvement in the sensitivity of SAW gas chromatography detection compared to the use of normal temperature or high temperature saturation for sample pretreatment. The detection time of the sample is only 2 minutes.

Example 6

The present embodiment detects the bergamot, including the following steps:

(1) Retention index calibration: In this embodiment, the retention index is used to formulate a digital fingerprint, which can effectively avoid errors caused by differences in operating factors and experimental conditions. Take 20 mg of 13 kinds of n-alkane mixed standard (C6-C18), put it into a 40 ml headspace sample bottle, use a solid-phase microextraction needle (in this example, use fused silica optical fiber) to extract for 20s, then the solid-phase microextraction needle It is connected with the port of the portable surface acoustic wave gas chromatograph, and the gas to be tested in the solid phase microextraction needle is sucked into the chromatograph through the pump and separated on the chromatographic column. The detection is carried out, and the detection signal is obtained on the surface acoustic wave detector, thereby obtaining and recording the peak time of each n-alkane standard product. The retention time of the normal alkane standard product peak is used to calculate the retention index of the volatile components of the sample to be tested using Kovats' retention index RI calculation formula in the subsequent step. The formula for calculating the retention index RI of Kovats is shown in formula (1).

(4) Standard product analysis experiment: buy bergamot from three different manufacturers, respectively numbered 10-1 bergamot, 10-2 bergamot and 10-3 bergamot, take 20mg of each of the above three bergamot as the standard, add 40ml headspace bottle Inside, after using the solid-phase microextraction needle to extract for 20s, connect the solid-phase microextraction needle to the portable surface acoustic wave gas chromatograph, and the gas to be tested is sucked into the chromatograph by the pump, separated on the chromatographic column, and detected by the surface acoustic wave The detection signal is obtained on the device. After the detection signal is obtained, the data processing device can calculate the retention index of the active ingredient in the bergamot standard according to the peak retention time of the normal paraffin standard product calibrated in step (1) and the retention time of the active ingredient in the bergamot standard. The data processing unit can also calculate the response (peak area) of the active ingredient in the bergamot standard. According to the sample peak retention index and response obtained in this example, a database of traditional Chinese medicine bergamot fingerprints was established. Among them, the response, that is, the peak area, can reflect the content of the corresponding active ingredient.

(5) Analysis experiment of samples of traditional Chinese medicines to be tested: In this example, a fake bergamot (fake bergamot) was used as the samples of traditional Chinese medicines to be tested. Take 20 mg of fake bergamot (fake bergamot), add it into a 40ml headspace bottle, use a solid-phase microextraction device to extract for 20s, and connect it to a portable surface acoustic wave gas chromatograph. separation, and the detection signal is obtained on the surface acoustic wave detector. After obtaining the detection signal, the data processing device can calculate the retention index of the detected active ingredient in bergamot according to the retention time of the normal alkane standard product calibrated in step (1) and the retention time of the detected active ingredient in bergamot. The data processing unit can also calculate the response (peak area) of the active ingredient in the bergamot standard. According to the comparison between the obtained sample peak retention index and the Chinese medicine bergamot fingerprint database established in step (4), the authenticity is identified by observing the similarity of the position and relative size of the spectral peaks of the main components.

13 shows the chromatogram of each bergamot detected in step (5). Figure 14 shows the chromatogram of 5-1 Bergamot and the retention index and response value of each peak. From the comparison of each spectrum in the figure, it can be clearly identified that the TCM samples to be tested are fake bergamot. The experimental results of this embodiment demonstrate that the method of the present invention can accurately detect and identify whether the Chinese medicine sample to be tested is genuine or counterfeit.

To sum up, the present invention provides a method for identifying traditional Chinese medicines by using a surface acoustic wave gas chromatograph. Compared with the prior art, the method can effectively eliminate the uncertainty caused by factors such as geographical environment, climatic conditions, weather conditions and instrument status in the field detection, and has the advantages of simple sample pretreatment, fast analysis speed, and field detection.

Claims

A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph, characterized in that it comprises the following steps:

(1) Determination of sample volatility: take the standard of the traditional Chinese medicine sample to be tested and carry out saturation treatment at room temperature to obtain the sample to be determined; use a surface acoustic wave gas chromatograph to detect the sample to be determined to obtain the volatility of the sample to be determined. The spectrum of the components; according to the response value of the largest peak of the response value, it is judged whether the sample to be tested is a highly volatile sample. The standard for determining the volatility of the sample is: the response value of the largest peak of the response value in the spectrum is greater than or equal to 2000Hz, which means high volatility sample;

(2) Sample pretreatment: after determining that the sample to be tested is a highly volatile sample according to step (1), the sample of the traditional Chinese medicine to be tested and its corresponding standard product are pretreated to obtain the sample to be tested and the standard sample. The pretreatment conditions For normal temperature saturation treatment;

(3) Obtaining the standard fingerprint: using a surface acoustic wave gas chromatograph to detect the standard sample to obtain the standard fingerprint of the volatile components in the standard;

(4) Detection of the sample: use a surface acoustic wave gas chromatograph to detect the sample to be tested, obtain a sample spectrum of volatile components in the traditional Chinese medicine sample to be tested, and compare the sample spectrum with the standard fingerprint spectrum obtained in step (3), Identify the authenticity of the Chinese medicine to be tested.
A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph according to claim 1, wherein: in the step (1) or step (2), the specific process of the normal temperature saturation treatment is, Take 20-1000mg samples of traditional Chinese medicine and put them into 10-40ml headspace vials, and saturate them for 1-60min at room temperature.
A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph according to claim 1, characterized in that: in the step (1), step (3) or step (4), the acoustic surface The test process of the wave gas chromatograph is as follows: in the sample injection state, the sampling port is connected to the sample to be tested, and the sampling pump is started, and the sample to be tested is sucked into the pre-concentration tube; in the detection state, the six-way valve is switched to make the carrier gas flow through Pre-concentration tube, and carry the sample to be tested in the pre-concentration tube into the chromatographic column and the detector in turn to complete the detection.
A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph according to claim 1 or 3, wherein the surface acoustic wave gas chromatographic column is selected from the group consisting of DB-5, SPB-5, Rtx-5, BP-5, OV-5, 007-2 (MPS-5), SE-52, SE-54, XTI-5, PTE-5, ZB-5, AT-5, MDN-5 or ZB -5, the length of the chromatographic column is 1-10 meters.
A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph according to claim 4, characterized in that: the detection conditions of the surface acoustic wave gas chromatography are as follows: the initial temperature of the chromatographic column is 40- 50°C, during the detection process, the temperature of the chromatographic column is raised to 200°C according to a temperature program of 0-20°C/s; the flow rate of the chromatographic column is 1-7mL/min, and the carrier gas is nitrogen or helium gas.
A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph according to claim 1 or 3, characterized in that: in the sampling state of the surface acoustic wave gas chromatograph, the sampling pump The working time is 5-60s.
A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph according to claim 1 or 3, characterized in that: the detection conditions of the surface acoustic wave gas chromatograph are as follows: The temperature is 25-60°C.
A method for on-site identification of highly volatile Chinese medicinal materials using a surface acoustic wave gas chromatograph according to claim 1, characterized in that: in step (4), using similarity evaluation or principal component analysis method, the sample atlas Compare with the standard fingerprint spectrum obtained in step (3) to identify the authenticity of the Chinese medicine to be tested.