A method for detecting illegal additives of cordyceps sinensis

Abstract

The application discloses a detection method of illegal additives of Cordyceps sinensis, and belongs to the technical field of traditional Chinese medicinal material detection, and comprises the steps of detecting illegal additives in Cordyceps sinensis. The illegal additives are at least one of perfluorotributylamine of formula I, perfluoropropyl vinyl ether of formula II, hexafluoropropylene oxide trimer of formula III and 2-perfluoropropoxy perfluoropropyl trifluorovinyl ether of formula IV.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing technology, and in particular to a method for detecting illegal additives in Cordyceps sinensis. Background Technology

[0002] Cordyceps sinensis is a unique Chinese medicinal herb, considered one of the three major tonics alongside ginseng and deer antler, and is recorded in ancient Chinese medical texts. Cordyceps sinensis has a sweet taste and neutral properties. It tonifies the kidneys and lungs, stops bleeding, and resolves phlegm. It can be used for kidney deficiency and spermatorrhea, impotence, seminal emission, lower back and knee pain, chronic cough and wheezing, and cough with hemoptysis.

[0003] However, Cordyceps sinensis still mainly relies on natural harvesting. Due to the continuous harvesting of Cordyceps sinensis resources, grasslands have been severely degraded, the grassland ecological environment has gradually deteriorated, and the quantity of Cordyceps sinensis has been declining. This has led to high market prices for Cordyceps sinensis, making it a high-value Chinese medicinal material.

[0004] During routine supervision, it was discovered that some Cordyceps sinensis was illegally treated with organic fluoride to increase its weight. To address this issue, the "fluoride test method" in the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Technical Requirements 0805, can be used. A blue-purple color is considered positive. However, the colorimetric reaction method is prone to false negatives due to insufficient sensitivity. Summary of the Invention

[0005] To address the problem of insufficient sensitivity in detecting illegally added organofluorine compounds in Cordyceps sinensis in the existing technologies mentioned above, this invention provides a method for detecting illegally added substances in Cordyceps sinensis, which clarifies the types of illegally added substances and is based on high-resolution gas chromatography-mass spectrometry (GC-MS) technology. This method has high sensitivity and can achieve quantitative detection.

[0006] This invention provides a method for detecting illegal additives in Cordyceps sinensis, including the step of detecting illegal additives in Cordyceps sinensis, wherein the illegal additives are at least one selected from the following formulas: perfluorotributylamine of Formula I, perfluoropropyl vinyl ether of Formula II, hexafluoropropylene oxide trimer of Formula III, and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether of Formula IV.

[0007]

[0008] During routine supervision, the inventors discovered that commercially available Cordyceps sinensis was illegally adulterated with organofluorine compounds. The standard method for testing fluoride is the "Fluoride Test" (0805) in the 2020 edition of the Chinese Pharmacopoeia, Part IV. However, this colorimetric reaction method lacks sensitivity and is prone to false negatives. Furthermore, due to limited current information about the true composition of these illegal additives, it is impossible to develop a more targeted, sensitive, and accurate detection method.

[0009] Accordingly, the inventors consulted a large number of relevant literature studies, but none yielded a more sensitive and specific feasible detection method, necessitating independent development. After exploration, mass spectrometry was chosen as the detection method, and combined with the properties of organofluorine compounds, gas chromatography-mass spectrometry (GC-MS) was used for structural confirmation. Initially, the database built into the GC-MS instrument was used for qualitative analysis, but the specific compound could not be identified, only a series of possible fluorine-containing or non-fluorine-containing compounds were identified. Further in-depth research revealed that different positive samples exhibited a series of identical fragment ions in their mass spectra. Simultaneously, screening for commercially available additives that met the requirements for illegal addition was conducted. Considering properties such as the need for colorless and odorless organic fluorine-containing liquids, it was speculated that the illegal additives might be perfluorotributylamine, perfluoropropyl vinyl ether, hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, etc. Experimental verification showed that these compounds exhibited a series of identical fragment ions in the GC-MS analysis using this method, and chromatographic peaks with corresponding retention times were extracted, indicating that the above substances are indeed the illegally added organofluorine compounds in Cordyceps sinensis.

[0010] In some of these methods, the method for detecting illegal additives in Cordyceps sinensis is gas chromatography-mass spectrometry (GC-MS), preferably high-resolution mass spectrometry (HDMS). GC-MS, particularly high-resolution GC-MS, can better distinguish between perfluoropropyl vinyl ether and hexafluoropropylene oxide trimer.

[0011] In some of these schemes, the detection method includes the following steps:

[0012] Preparation of test sample: Take the Cordyceps sinensis sample to be tested, add organic solvent to extract, and obtain test sample solution;

[0013] Chromatographic separation: Using a capillary column with a weakly polar arylene stationary phase as the stationary phase, the temperature was increased from 30-40℃ to 220-240℃ in a gradient and held for 5-15 min for gas chromatography separation.

[0014] Mass spectrometry detection: In positive ion mode, fragment ion chromatographic peaks at m / z 219±0.1, 266±0.1, 267±0.1, and 335±0.1 were detected by mass spectrometry. Among them, m / z 219±0.1 is the characteristic fragment ion of perfluorotributylamine, m / z 266±0.1 is the characteristic fragment ion of perfluoropropyl vinyl ether, m / z 267±0.1 is the characteristic fragment ion of hexafluoropropylene oxide trimer, and m / z 335±0.1 is the characteristic fragment ion of hexafluoropropylene oxide trimer and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether.

[0015] In some of these schemes, the mass spectrometry detection step further includes detecting fragment ion chromatographic peaks with m / z values ​​of 285±0.1, 169±0.1, 147±0.1, 119±0.1, 97±0.1, and 69±0.1. If no fragment ion with m / z 335±0.1 is present, but this series of fragment ion chromatographic peaks are present, it can be inferred that an illicit additive of the same type as hexafluoropropylene oxide trimer and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether (PFTP) is present.

[0016] In some of these solutions, the organic solvent is an acetone-n-heptane mixture with a volume ratio of 0.5-1.5:1, preferably a acetone-n-heptane mixture with a volume ratio of 1:1. Using this mixed solvent, perfluorotributylamine, perfluoropropyl vinyl ether, hexafluoropropylene oxide trimer, and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether can be dissolved relatively well.

[0017] In some of these methods, the extraction is performed by ultrasonic extraction at 0-50°C for 20-40 minutes, with an ultrasonic power of 25-45 kHz. Preferably, the extraction is performed by ultrasonic extraction at 20-30°C for 30 minutes, with an ultrasonic power of 37 kHz.

[0018] In some of these schemes, in the test sample preparation step, the test sample solution is prepared by extracting 0.1 ± 0.02 g of the Cordyceps sinensis sample to be tested per milliliter of organic solvent. Preferably, the test sample solution is prepared by extracting 0.1 g of the Cordyceps sinensis sample to be tested per milliliter of organic solvent.

[0019] In the chromatographic separation step, the injection volume is 0.5-2 μl, preferably 1 μl.

[0020] The amounts selected for preparing the above-mentioned test solution were calculated by the inventors based on the addition range of most illegal additives after extensive research, sample collection and testing. Preparing the test solution using the above method can yield suitable detection peaks, which is beneficial for subsequent quantitative analysis.

[0021] In some of these designs, the capillary column is a TG-5 SILMS column. Preferably, the capillary column has the following specifications: 30m × 0.25mm, 0.25μm. That is, a length of 30 meters, a diameter of 0.25mm, and a liquid film thickness of 0.25μm.

[0022] In some of these schemes, the temperature conditions for the chromatographic separation step are as follows: the initial temperature is 35±2℃, and the temperature is uniformly increased to 230±5℃ at a rate of 10±1℃, and held for 10±3 min; preferably, the initial temperature is 35℃, and the temperature is uniformly increased to 230℃ at a rate of 10℃ / min, and held for 10 min.

[0023] In some of these schemes, the injection port temperature during the chromatographic separation step is 220-260°C, preferably 250°C.

[0024] In some of these schemes, the carrier gas in the chromatographic separation step is an inert gas, preferably nitrogen. In some of these schemes, the split ratio in the chromatographic separation step is 80-120:1, preferably 100:1.

[0025] In some of these schemes, the conditions for mass spectrometry detection are: ion source: electron impact ion source;

[0026] In some of these schemes, the conditions for mass spectrometry detection are: ion source temperature: 250±20℃, preferably 250℃.

[0027] In some of these schemes, the conditions for mass spectrometry detection are: electron bombardment energy: 70 ± 5 eV, preferably 70 eV.

[0028] In some of these schemes, the conditions for mass spectrometry detection are: transmission line temperature: 250±20℃, preferably 250℃.

[0029] In some of these schemes, the detection method further includes a chromatographic peak analysis step, which is as follows:

[0030] If a fragment ion chromatographic peak with an m / z of 219±0.1 is obtained by mass spectrometry, it is determined that perfluorotributylamine has been added to the Cordyceps sinensis sample to be tested.

[0031] If a fragment ion chromatographic peak with an m / z of 267±0.1 is obtained by mass spectrometry, it is determined that the Cordyceps sinensis sample to be tested contains hexafluoropropylene oxide trimer.

[0032] When a fragment ion chromatographic peak with an m / z of 266±0.1 is detected by mass spectrometry, it is determined that perfluoropropyl vinyl ether has been added to the Cordyceps sinensis sample to be tested.

[0033] If mass spectrometry detection yields only a fragment ion chromatographic peak with m / z 335±0.1 and no fragment ion chromatographic peak with m / z 267±0.1, it is determined that the Cordyceps sinensis sample to be tested contains 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether.

[0034] When mass spectrometry detects fragment ion peaks at m / z 335±0.1 and m / z 267±0.1, it is determined that the Cordyceps sinensis sample contains 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether or hexafluoropropylene oxide trimer and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether. Based on common knowledge in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain various preferred embodiments of the present invention.

[0035] The reagents and raw materials used in this invention are all commercially available.

[0036] The positive and progressive effects of this invention are as follows:

[0037] The present invention discloses a method for detecting illegal additives in Cordyceps sinensis. By analyzing and identifying the specific types of illegally added organofluorine compounds in Cordyceps sinensis, the method ultimately identifies one or more of the following: perfluorotributylamine, perfluoropropyl vinyl ether, hexafluoropropylene oxide trimer, and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether.

[0038] This detection method can perform targeted qualitative or quantitative detection of specific illegal additives, thereby improving detection sensitivity and stability and avoiding false negative results. Attached Figure Description

[0039] Figure 1 The graph shows the detection results of perfluorotributylamine reference standard, where: A is the mass chromatogram and B is the mass bar graph.

[0040] Figure 2 The graph shows the detection results of hexafluoropropylene oxide trimer reference standard, where: A is the mass chromatogram and B is the mass bar graph.

[0041] Figure 3 The graph shows the detection results of 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether reference standard, where: A is the mass chromatogram and B is the mass bar graph.

[0042] Figure 4 The graph shows the detection results of perfluoropropyl vinyl ether reference standard, where: A is the mass chromatogram and B is the mass bar graph.

[0043] Figure 5 The chromatograms are mass chromatograms of the spiked sample solutions, where: A is the chromatogram for m / z 266, 267, and 335, and B is the chromatogram for m / z 219.

[0044] Figure 6 This is a bar graph showing the mass of samples with m / z 266, 219, 267, and 335 in the spiked sample solution.

[0045] Figure 7The chromatograms are for the blank solvent acetone-n-heptane (1:1), where: A is the chromatogram for m / z 266, 267, and 335, and B is the chromatogram for m / z 219.

[0046] Figure 8 The chromatograms are for the negative sample solution (number HC-2), where: A is the chromatogram for m / z 266, 267, and 335, and B is the chromatogram for m / z 219.

[0047] Figure 9 Mass chromatograms of HC-4 sample at m / z 266, 335, and 267 (0–1.65 min) were obtained.

[0048] Figure 10 This is a bar graph of the mass at 1.53 min in sample HC-4.

[0049] Figure 11 Mass chromatogram of sample HC-4 at m / z 219 (1.9–3.3 min).

[0050] Figure 12 This is a bar graph of the mass of sample HC-4 at 2.00 min.

[0051] Figure 13 Mass chromatograms of HC-4 samples at m / z 266, 335, 267, and 219 were obtained (4–34.5 min).

[0052] Figure 14 This is a bar graph of the mass at 4.33 min in sample HC-4.

[0053] Figure 15 Mass chromatogram of sample YS-1 at m / z 219 (1.9–3.3 min).

[0054] Figure 16 This is a bar graph of the mass at 2.62 min in sample YS-1.

[0055] Figure 17 Mass chromatograms of sample YS-1 at m / z 266, 335, 267, and 219 were obtained (4–34.5 min).

[0056] Figure 18 This is a bar graph of the mass at 9.70 min in sample YS-1.

[0057] Figure 19 Mass chromatograms of sample RG-1 at m / z 266, 335, 267, and 219 were obtained (4–34.5 min).

[0058] Figure 20This is a bar graph of the mass at 11.78 min in sample RG-1.

[0059] Figure 21 The colorimetric reaction results are from the "fluoride test" in Comparative Example 1.

[0060] Figure 22 The colorimetric reaction results of different proportions of perfluorinated compounds were examined using the "fluorine test method" in Comparative Example 2.

[0061] Figure 23 The mass chromatograms of the mixture in Comparative Example 2 with a blending ratio of 0.05% are shown below. In Comparative Example 2, A is the chromatogram of m / z 266, 267, and 335, and B is the chromatogram of m / z 219.

[0062] Figure 24 The mass chromatograms of the mixture in Comparative Example 2 with a blending ratio of 0.1% are shown below. In Comparative Example 2, A is the chromatogram of m / z 266, 267, and 335, and B is the chromatogram of m / z 219.

[0063] Figure 25 The mass chromatogram for Comparative Example 2 with a blending ratio of 1% is shown, where: A is m / z

[0064] Chromatograms of 266, 267, and 335, and chromatogram of B at m / z 219. Detailed Implementation

[0065] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0066] Instruments: Thermo Exactive GC high-resolution gas chromatography-mass spectrometry; Mettler Toledo 0.0 ...

[0067] Materials: Perfluorotributylamine (lot no: LRAD1370, purity 99.5%, purchased from SIGMA-ALDRICH), hexafluoropropylene oxide trimer (lot: A2218271, purity ≥95%, purchased from Aladdin), 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether (lot: C15914470, purity ≥98%, purchased from Maclean's), perfluoropropylvinyl ether (lot: PRJCL-00, purity >98.0%, purchased from TCI). Some of the collected Cordyceps sinensis samples are shown in Table 1.

[0068] Table 1 Sample Information Table

[0069]

[0070]

[0071] Example 1

[0072] A method for detecting illegal additives in Cordyceps sinensis includes the following steps:

[0073] 1. Preparation of test sample

[0074] Take an appropriate amount of Cordyceps sinensis, crush it, weigh about 0.5g, accurately add 5ml of a 1:1 mixture of acetone and n-heptane, sonicate for 30 minutes, shake well, filter, and take the filtrate to obtain the test solution.

[0075] 2. Preparation of reference standards

[0076] Take appropriate amounts of perfluorotributylamine, hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, and perfluoropropylvinyl ether reference standards, and add acetone-n-heptane (1:1) to prepare reference solutions with concentrations of 17 μg / ml, 100 μg / ml, 15 μg / ml, and 13 μg / ml, respectively.

[0077] 3. Chromatographic separation

[0078] The chromatographic conditions are as follows:

[0079] Column: TG-5SILMS (Thermo Fisher Scientific: 30m × 0.25mm, 0.25μm).

[0080] The column temperature is programmed: starting at 35°C, hold for 5 minutes, then increase to 230°C at a rate of 10°C per minute, and hold for 10 minutes.

[0081] Inlet temperature: 250℃.

[0082] Injection method and volume: 1 μl of solution was directly injected.

[0083] Carrier gas: nitrogen.

[0084] Split ratio: 100:1.

[0085] 4. Mass spectrometry detection

[0086] In positive ion mode, the chromatographic peaks at m / z 219, 266, 267, and 335 were detected by mass spectrometry. Among them, m / z 219 is the characteristic fragment ion of perfluorotributylamine, m / z 266 is the characteristic fragment ion of perfluoropropyl vinyl ether, m / z 267 is the characteristic fragment ion of hexafluoropropylene oxide trimer, and m / z 335 is the characteristic fragment ion of fluoropropylene oxide trimer and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether.

[0087] The mass spectrometry detection conditions are as follows:

[0088] Ion source: Electron impact ion source (EI), positive ion mode.

[0089] Ion source temperature: 250℃.

[0090] Electron bombardment energy: 70 eV.

[0091] Transmission line temperature: 250℃.

[0092] Quality scan range: m / z 33~700.

[0093] Example 2

[0094] Verification of the detection method in Example 1.

[0095] 1. Method

[0096] The test was performed according to the method in Example 1, with additional spiked samples added.

[0097] Preparation of spiked sample solution: Take an appropriate amount of Cordyceps sinensis (verified to be free of illegal organic fluoride additives), crush it, weigh about 0.5g accurately, add 5ml of acetone-n-heptane mixed solvent with a volume ratio of 1:1 (containing approximately 100μg / ml each of perfluorotributylamine, hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, and perfluoropropylvinyl ether), sonicate for 30 minutes, shake well, filter, and collect the filtrate to obtain the spiked sample solution.

[0098] 2. Results and Analysis

[0099] 2.1 Determination of reference solution, spiked sample solution and blank solvent

[0100] (1) Detection results and analysis of reference solution

[0101] Gas chromatograms of reference standards for perfluorotributylamine (PFTBA), hexafluoropropylene oxide trimer (HFPO-TA), 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether (PTE), and perfluoropropyl vinyl ether (PPVE) are shown below. Figure 1-4 As shown.

[0102] in, Figure 1 A represents the mass chromatogram of the perfluorotributylamine fragment ion at m / z 218.985. Figure 1 Figure B shows the mass bar graph of perfluorotributylamine when the response signal value is highest at a retention time of 2.0 min.

[0103] Figure 2 A represents the mass chromatogram of the hexafluoropropylene oxide trimer fragment ion at m / z 266.986. Figure 2Figure B shows the mass bar graph of hexafluoropropylene oxide trimer at the highest response signal value with a retention time of 1.57 min.

[0104] Figure 3 A represents the mass chromatogram of the fragment ion of 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether at m / z 334.973. Figure 3 Figure B shows the mass bar graph of 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether when the response signal value is highest at a retention time of 1.53 min.

[0105] Figure 4 A in the image is a mass chromatogram of the perfluoropropyl vinyl ether fragment ion at m / z 265.979. Figure 4 Figure B shows the mass bar graph of perfluoropropyl vinyl ether at the highest response signal value with a retention time of 1.45 min.

[0106] Analysis of the results shows that... Figure 1 Perfluorotributylamine produces fragment ions with m / z 69, 131, and 219. Among them, m / z 219 can be distinguished from the other three compounds, thus m / z 219 is identified as the characteristic fragment ion of perfluorotributylamine.

[0107] Figure 2 The hexafluoropropylene oxide trimer produces fragment ions with m / z values ​​of 69, 147, and 267. Among them, m / z 267 can be distinguished from the other three compounds, thus m / z 267 is identified as the characteristic fragment ion of the hexafluoropropylene oxide trimer.

[0108] Figure 3 2-Perfluoropropoxyperfluoropropyltrifluorovinyl ether produces fragment ions with m / z 69, 147, and 335. Due to the low specificity of the obtained fragment structures, no characteristic peaks that are exclusive to and completely distinguishable from other fluorinated organic compounds were obtained. Considering that m / z 335 has a certain degree of specificity, m / z 335 is selected as the characteristic fragment ion of 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether.

[0109] Figure 4 Perfluoropropyl vinyl ether produces fragment ions with m / z 69, 147, and 266. Among them, m / z 266 can be distinguished from the other three compounds, thus m / z 266 is identified as the characteristic fragment ion of perfluoropropyl vinyl ether.

[0110] The structures corresponding to the fragment ions of the above-mentioned fluorinated organic compounds are shown in the table below.

[0111] Table 2. Fragment ions and structures of various fluorinated organic compounds

[0112]

[0113]

[0114] The fragment peaks and structures of each fluorinated organic compound are as described above. Among them, perfluorotributylamine, perfluoropropyl vinyl ether and hexafluoropropylene oxide trimer all have highly specific fragments with m / z values ​​of 219, 266 and 267, respectively.

[0115] For 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, there is no absolutely specific fragment peak. Therefore, m / z 335 is considered as its characteristic peak. However, since the characteristic peak of 335 also exists in the hexafluoropropylene oxide trimer, the judgment criteria are set as follows: if only m / z 335 is detected, it is considered that 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether has been added; if m / z 335 and m / z 267 are detected, it is considered that hexafluoropropylene oxide trimer has been added. Alternatively, if both 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether and hexafluoropropylene oxide trimer have been added, further structural analysis can be performed for confirmation.

[0116] (2) Detection results and analysis of spiked sample solutions

[0117] The mass spectrum of the spiked sample solution (approximately 100 μg / ml each of perfluorotributylamine, hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, and perfluoropropylvinyl ether) is shown below. Figure 5-6 As shown.

[0118] in, Figure 5 Mass chromatograms of fragment ions extracted from spiked sample solutions at m / z 219, 267, 335, and 266, respectively. Figure 6 This is a mass bar graph showing the peak signal intensity at each mass-to-charge ratio chromatogram.

[0119] The results showed that the corresponding chromatographic peaks were detected in the mass chromatograms of fragment ions extracted at m / z 219, 267, 335, and 266, indicating that the method can effectively detect perfluorotributylamine, hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, and perfluoropropylvinyl ether in the sample.

[0120] (3) Blank solvent determination results and analysis

[0121] The test was performed using acetone-n-heptane (1:1) solvent as a blank control, and the results are as follows: Figure 7 As shown, Figure 7 Mass chromatograms of fragment ions extracted at m / z 219, 267, 335, and 266 for the blank control.

[0122] The results showed that no corresponding chromatographic peaks were detected, indicating that the blank solvent did not interfere with the method.

[0123] (4) Sample solution detection results and analysis

[0124] The seven batches of samples collected (numbered: HC-1, HC-2, HC-3, HC-4, RG-1, YS-1, JP-1) were measured (concentration of 0.1 g / ml).

[0125] The results showed that samples numbered HC-1, HC-2, HC-3, JP-1, RG-1, and YS-1 did not exhibit any chromatographic peaks corresponding to the retention times of perfluorotributylamine, hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, and perfluoropropyl vinyl ether reference standards within the range of 0–3 min. The chromatogram of the exemplary sample HC-2 is shown below. Figure 8 As shown.

[0126] Sample HC-4 showed chromatographic peaks within 0–3 min corresponding to the retention times of 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether and perfluorotributylamine reference standards, such as… Figure 9-12 As shown.

[0127] in, Figure 9 The mass chromatogram of fragment ions with m / z 266, 335, and 267 extracted from sample HC-4 in the range of 0–1.65 min is shown in the figure. The fragment ion with m / z 335 and no m / z 267 has a chromatographic peak corresponding to the reference standard at a retention time of 1.53 min, indicating that 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether has been added. Figure 10 The bar graph shows the mass of sample HC-4 at 1.53 min (the retention time corresponding to the corresponding control). Figure 11 The mass chromatogram of the m / z 219 fragment ion extracted from sample HC-4 (1.9–3.3 min) shows a chromatographic peak with m / z 219 fragment ion, and a chromatographic peak corresponding to the reference standard at a retention time of 2.0 min, indicating that perfluorotributylamine has been added. Figure 12 This is a bar graph of the mass of sample HC-4 at 2.00 min.

[0128] Samples numbered HC-4, YS-1, and RG-1 all showed a series of chromatographic peaks containing fragment ions at m / z 69, 97, 119, 147, 169, 285, and 335 within 3.8-18 min. This series of peaks was extremely similar to the reference standards for hexafluoropropylene oxide trimer and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, and the number of peaks was high. Preliminary judgment suggests they are polymers of hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, or similar compounds. Figure 13-20 As shown.

[0129] in, Figure 13 Mass chromatograms of fragment ions at m / z 266, 335, 267, and 219 extracted from sample HC-4 (4–34.5 min). Figure 14 This is a bar graph of the mass at 4.33 min in sample HC-4.

[0130] Figure 15 Mass chromatogram of m / z 219 fragment ions extracted from sample YS-1 (1.9–3.3 min). Figure 16 This is a bar graph of the mass at 2.62 min in sample YS-1. Figure 17 Mass chromatograms of fragment ions at m / z 266, 335, 267, and 219 extracted from sample YS-1 (4–34.5 min). Figure 18 This is a bar graph of the mass at 9.70 min in sample YS-1.

[0131] Figure 19 Mass chromatograms of fragment ions at m / z 266, 335, 267, and 219 were extracted from sample RG-1 (4–34.5 min). Figure 20 This is a bar graph of the mass at 11.78 min in sample RG-1.

[0132] Depend on Figures 13-20 It can be seen that, in addition to the chromatographic peaks corresponding to the reference standards 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether and perfluorotributylamine in the range of 0–3 min, sample HC-4 also showed a series of chromatographic peaks containing fragment ions of m / z 69, 97, 119, 147, 169, 285, and 335 in the range of 4–34.5 min. It is speculated that sample HC-4 may contain other similar fluorinated organic compounds or polymers of the same type. Samples YS-1 and RG-1 did not show the chromatographic peaks corresponding to the above four reference standards such as perfluorotributylamine in the range of 0–3 min, indicating that the above four fluorinated organic compounds were not added. However, a series of chromatographic peaks containing fragment ions of m / z 69, 97, 119, 147, 169, 285, and 335 were detected in samples YS-1 and RG-1 in the range of 4–34.5 min. It is speculated that samples YS-1 and RG-1 may contain other similar fluorinated organic compounds or polymers of the same type.

[0133] The above results indicate that the method of this embodiment can not only effectively detect illegally added perfluorotributylamine, hexafluoropropylene oxide trimer, 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether and perfluoropropylvinyl ether in Cordyceps sinensis samples, but also indicate that other similar, same type of fluorinated organic compounds or polymers may have been added to the sample. The method is specific and there is no interference from blanks.

[0134] Example 3

[0135] This embodiment follows the method of Example 1, but the selected extraction solvents are methanol and n-heptane, respectively. The results showed that the compatibility of these two solvents with perfluorotributylamine, hexafluoropropylene oxide trimer, and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether reference standards was not as good as that of the acetone-n-heptane mixed solvent at a volume ratio of 1:1.

[0136] Comparative Example 1

[0137] This comparative example uses a colorimetric method to detect the collected samples.

[0138] 1. Principle

[0139] Organic drugs containing fluorine in their molecules are burned in an oxygen-filled combustion flask. After the combustion products are absorbed into the absorbent, they are tested according to the "Fluorine Test Method" in Part IV, General Technical Requirements 0805 of the 2020 edition of the Chinese Pharmacopoeia. A blue-purple color indicates a positive result.

[0140] 2. Method

[0141] 1) Preparation of test solution: Take about 50 mg of sample powder and destroy the organic matter by oxygen flask combustion method (General Rule 0703). Use 20 ml of water and 6.5 ml of 0.01 mol / L sodium hydroxide solution as the absorption liquid. After combustion is complete, shake thoroughly to obtain the test solution.

[0142] 2) Preparation of blank solution: Without adding the test sample, follow the steps above to prepare a blank solution.

[0143] 3) Determination: Take 2 ml of blank solution and test solution and place them in 25 ml colorimetric tubes respectively. Add 0.5 ml of alizarin fluorescein test solution, then add 0.2 ml of 12% sodium acetate dilute acetic acid solution, dilute with water to 4 ml, add 0.5 ml of cerium nitrate test solution, shake well, and carry out the color reaction.

[0144] 4) Results:

[0145] The results of the colorimetric reaction are shown in the figure. Figure 21 The results showed that the blank solution tube, HC-1, HC-2, HC-3, JP-1, and RG-1 did not show a blue-purple color, while HC-4 and YS-1 both showed a blue-purple color. Therefore, it was determined that samples HC-4 and YS-1 contained organic fluorides. The experimental results are compared with those of Example 2 in the table below.

[0146] Table 3. Comparison of results between colorimetric reaction method and high-resolution gas chromatography-mass spectrometry.

[0147]

[0148]

[0149] The results showed that the illegal additives could be detected in sample RG-1 using the detection method in Example 2, but could not be detected using the colorimetric method, indicating that the colorimetric reaction method is prone to false negative results due to different sensitivities.

[0150] Comparative Example 2

[0151] This comparative experiment compares the colorimetric reactions and gas chromatography-mass spectrometry (GC-MS) analysis results of different proportions of perfluorinated compounds (0.1%, 0.5%, 1%, and 10%, respectively).

[0152] 1. Method

[0153] The tests were performed using the methods described in Comparative Example 1 and Example 2.

[0154] 2. Results

[0155] The results of the colorimetric reaction are as follows Figure 22 As shown, the results indicate that samples with a perfluorinated compound content of 1% or less are not sensitive enough for color development, which can easily lead to misjudgment.

[0156] Gas chromatography-mass spectrometry analysis results are as follows Figure 23-25 As shown, the results indicate that each compound can be sensitively detected when the proportions of perfluorinated compounds are 0.05%, 0.1%, and 1%.

[0157] The above results indicate that gas chromatography-mass spectrometry is more sensitive than colorimetric reactions for detecting perfluorinated compounds in Cordyceps sinensis.

Claims

1. A method for detecting illegal additives in Cordyceps sinensis, characterized in that, The method includes a step of detecting illegal additives in Cordyceps sinensis, wherein the illegal additives are at least one of the following: perfluorotributylamine of Formula I, perfluoropropyl vinyl ether of Formula II, hexafluoropropylene oxide trimer of Formula III, and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether of Formula IV. ； ； The method for detecting illegal additives in Cordyceps sinensis includes the following steps: Preparation of test sample: Take the Cordyceps sinensis sample to be tested, add organic solvent for extraction, and obtain test sample solution; the organic solvent is a mixture of acetone and n-heptane with a volume ratio of 0.5-1.5:

1. Chromatographic separation: Using a capillary column with a weakly polar arylene stationary phase as the stationary phase, the temperature was increased from 30-40℃ to 220-240℃ in a gradient and held for 5-15 min for gas chromatography separation. Mass spectrometry detection: In positive ion mode, fragment ion chromatographic peaks with m / z of 219±0.1, 266±0.1, 267±0.1, and 335±0.1 were detected by mass spectrometry. Among them, m / z 219±0.1 is the characteristic fragment ion of perfluorotributylamine, m / z 266±0.1 is the characteristic fragment ion of perfluoropropyl vinyl ether, m / z 267±0.1 is the characteristic fragment ion of hexafluoropropylene oxide trimer, and m / z 335±0.1 is the characteristic fragment ion of hexafluoropropylene oxide trimer and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether. It also includes a chromatographic peak analysis step, which is as follows: If a fragment ion chromatographic peak with an m / z of 219±0.1 is detected by mass spectrometry, it is determined that perfluorotributylamine has been added to the Cordyceps sinensis sample to be tested. If a fragment ion chromatographic peak with an m / z of 267±0.1 is obtained by mass spectrometry, it is determined that the Cordyceps sinensis sample to be tested contains hexafluoropropylene oxide trimer. When a fragment ion chromatographic peak with an m / z of 266±0.1 is detected by mass spectrometry, it is determined that perfluoropropyl vinyl ether has been added to the Cordyceps sinensis sample to be tested. If mass spectrometry detection yields only a fragment ion chromatographic peak with m / z 335±0.1 and no fragment ion chromatographic peak with m / z 267±0.1, it is determined that the Cordyceps sinensis sample to be tested contains 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether. If mass spectrometry detects fragment ion chromatographic peaks with m / z of 335±0.1 and m / z of 267±0.1, it is determined that the Cordyceps sinensis sample to be tested contains 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether, or hexafluoropropylene oxide trimer and 2-perfluoropropoxyperfluoropropyltrifluorovinyl ether.

2. The method for detecting illegal additives in Cordyceps sinensis according to claim 1, characterized in that, The mass spectrometry used is high-resolution mass spectrometry.

3. The method for detecting illegal additives in Cordyceps sinensis according to claim 1, characterized in that, The mass spectrometry detection step also includes detecting fragment ion chromatographic peaks with m / z values ​​of 285±0.1, 169±0.1, 147±0.1, 119±0.1, 97±0.1, and 69±0.

1.

4. The method for detecting illegal additives in Cordyceps sinensis according to claim 1, characterized in that, The organic solvent is an acetone-n-heptane mixed solvent with a volume ratio of 1:1; And / or, The extraction method is ultrasonic extraction at 0-50℃ for 20-40 min, and the ultrasonic power is 25-45 kHz.

5. The method for detecting illegal additives in Cordyceps sinensis according to claim 4, characterized in that, The extraction method involves ultrasonic extraction at 20-30℃ for 30 minutes, with an ultrasonic power of 37kHz.

6. The method for detecting illegal additives in Cordyceps sinensis according to claim 1, characterized in that, In the test sample preparation step, the test sample solution is prepared by extracting 0.1±0.02g of the Cordyceps sinensis sample to be tested per milliliter of organic solvent; In the chromatographic separation step, the injection volume is 0.5-2 μl.

7. The method for detecting illegal additives in Cordyceps sinensis according to claim 6, characterized in that, In the test sample preparation step, the test sample solution is prepared by extracting 0.1g of the Cordyceps sinensis sample to be tested per milliliter of organic solvent.

8. The method for detecting illegal additives in Cordyceps sinensis according to claim 6, characterized in that, In the chromatographic separation step, the injection volume is 1 μl.

9. The method for detecting illegal additives in Cordyceps sinensis according to any one of claims 1-8, characterized in that, The capillary column is a TG-5SILMS column.

10. The method for detecting illegal additives in Cordyceps sinensis according to claim 9, characterized in that, The capillary column has the following specifications: 30m × 0.25mm, 0.25μm.

11. The method for detecting illegal additives in Cordyceps sinensis according to claim 10, characterized in that, The temperature conditions for the chromatographic separation step are as follows: the initial temperature is 35±2℃, and the temperature is increased uniformly to 230±5℃ at a rate of 10±1℃, and held for 10±3min.

12. The method for detecting illegal additives in Cordyceps sinensis according to claim 11, characterized in that, The temperature conditions for the chromatographic separation step are as follows: the initial temperature is 35℃, and the temperature is increased to 230℃ at a rate of 10℃ / min and held for 10min.

13. The method for detecting illegal additives in Cordyceps sinensis according to any one of claims 1-8, characterized in that, The chromatographic separation step also includes at least one of the following conditions: (1) The injection port temperature is 220-260℃; (2) The carrier gas is an inert gas; (3) The split ratio is 80-120:1; And / or, The conditions for the mass spectrometry detection also include at least one of the following conditions: (1) Ion source: Electron bombardment ion source; (2) Ion source temperature: 250±20℃; (3) Electron bombardment energy: 70±5eV; (4) Transmission line temperature: 250±20℃.

14. The method for detecting illegal additives in Cordyceps sinensis according to claim 13, characterized in that, The chromatographic separation step also includes at least one of the following conditions: (1) The injection port temperature is 250℃; (2) The carrier gas is nitrogen; (3) The split ratio is 100:1; And / or, The conditions for the mass spectrometry detection also include at least one of the following conditions: (1) The ion source temperature is 250℃; (2) The electron bombardment energy is 70 eV; (3) The transmission line temperature is 250℃.

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