Quantitative analysis method for sulphitation marker peimisine sulfonate in fritillaria traditional Chinese medicinal materials

Abstract

The invention discloses a quantitative analysis method for a sulphitation marker peimisine sulfonate in fritillaria traditional Chinese medicinal materials. The method comprises the following steps: crushing and sieving to-be-detected fritillaria, sequentially adding a reference substance stock solution and a methanol solution of peimisine sulfonate, performing ultrasonic extraction, and filtering an extracting solution through a filter membrane to obtain filtrate; and carrying out ultra-high performance liquid chromatography-evaporative light scattering detection on the filtrate to realize the determination of the content of the peimisine sulfonate in the fritillary bulb to be detected. Chromatographic conditions of the ultra-high performance liquid chromatography-evaporative light scattering detection are as follows: a mobile phase is ultrapure water and acetonitrile in a volume ratio of 85: 15; the elution mode is isocratic elution; the chromatographic column is an ACQUITY # imgabs0 # HSS T3 chromatographic column; the flow velocity is 0.2 to 0.4 mL / min; the column temperature is 25-35 DEG C; the qualitative and quantitative detection method suitable for the sulfur-smoked fritillaria traditional Chinese medicinal materials is established based on the UPLC-ELSD technology, has the advantages of being low in cost, easy and convenient to operate, high in sensitivity and rapid, is wide in application range and has important practical application value, and the problem that an existing detection method is narrow in application range is solved to a certain extent.

Description

Technical Field

[0001] The invention relates to a quantitative analysis method of sulfur-fumigation marker fritillaria sulfonate in fritillaria-type traditional Chinese medicinal materials, and belongs to the technical field of analysis. Background Art

[0002] In recent years, the phenomenon of using sulfur fumigation or soaking sodium metabisulfite compounds on Chinese medicinal materials, food and agricultural products (hereinafter referred to as "sulfur fumigation") is common, and is commonly known as "sulfur treatment" in production. Sulfur fumigation was widely used in the 1970s and 1980s because of its advantages of drying, sterilization, insect prevention, anti-corrosion and mildew prevention, as well as easy storage. However, a large number of studies have shown that sulfur fumigation may cause significant changes in the types and contents of chemical components, thereby affecting the pharmacological effects of Chinese medicinal materials and the nutritional value of food. It may also lead to residues of sulfur dioxide (SO2) and heavy metals (such as arsenic, mercury, etc.), which may cause potential harm to human organs such as liver and kidneys.

[0003] To ensure the safety of food and medicine, my country has issued a number of relevant laws and technical standards since 2000. The Chinese Pharmacopoeia and the Food Additives Usage Standards also clearly stipulate the limit requirements for sulfur fumigation residues. However, since the sulfur fumigation method is simple, easy to use, low-cost, and has the effects of weight gain, bleaching, color enhancement and color retention, the appearance of the processed products is more attractive, misleading consumers to buy. Therefore, sulfur fumigation is still widely used in the processing, preparation and storage of Chinese medicinal materials and food ingredients at the place of origin, and has become one of the "unwritten rules" in production. This irregular operation not only disrupts the market order and damages the industry's reputation, but also poses hidden dangers to clinical drug use and food safety.

[0004] Monitoring the residual sulfur dioxide in Chinese medicinal materials is of great significance to ensure the safety and effectiveness of clinical drugs. Establishing a rapid and accurate detection technology system is one of the key means to achieve effective supervision and management. With the development of desulfurization technology for Chinese medicinal materials, the concealment of sulfur fumigation has gradually increased. Relying solely on the monitoring of SO2 residuals is no longer sufficient to fully reflect the degree of sulfur fumigation. How to achieve rapid and accurate detection of desulfurized Chinese medicinal materials has become a hot topic and difficulty in the industry for a long time. Studies have shown that sulfur fumigation of Chinese medicinal materials will not only reduce the quality of medicinal materials, but also generate some new sulfur-containing derivatives. These compounds can be used as important markers to determine whether Chinese medicinal materials have been treated with sulfur fumigation. Exploring stable and reliable sulfur fumigation markers, especially the specific sulfur-containing markers newly generated after sulfur fumigation, is the key to achieving quality control of sulfur fumigated Chinese medicinal materials. However, the current research on sulfur fumigation markers is mostly focused on a single Chinese medicinal material, and the developed quality markers have a narrow scope of application. There is still a lack of common sulfur fumigation markers that can be universally applied to a variety of Chinese medicinal materials.

[0005] Based on the basic principle of the formation of sulfur-fumigated markers of traditional Chinese medicine, that is, certain components or specific groups react with sulfite to form an addition reaction, the author conducted research on sulfur-fumigated Fritillaria thunbergii, Fritillaria cirrhosa, Fritillaria hupehensis, Fritillaria dasyphylla and Fritillaria chuanxiong, and found that the sulfur-fumigated Fritillaria thunbergii all generated a stable compound - Fritillaria sulfonate. This compound can be used as an important marker to determine whether Fritillaria thunbergii has been treated with sulfur fumigation. However, a quantitative analysis method for Fritillaria sulfonate has not yet been established, which to a certain extent limits its further development in the research and application of sulfur-fumigated markers. The present invention is specially proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a quantitative analysis method for Fritillaria sulfonate based on ultra-high performance liquid chromatography-evaporative light scattering detection technology (UPLC-ELSD), which provides technical support for rapid identification of Fritillaria sulfur-fumigated medicinal materials, helps to improve the quality control level of Fritillaria medicinal materials, and provides a new technical reference for the desulfurization and sulfur-fumigation marker research of Chinese medicinal materials.

[0007] The structural formula of the sulfonic acid fritillariae sulphonate of the present invention is shown in Formula I:

[0008]

[0009] The quantitative analysis method of sulfur-fumigation marker fritillaria sulfonate in fritillaria Chinese medicinal materials provided by the present invention comprises the following steps:

[0010] S1. The tested Fritillaria is crushed and sieved, and a reference substance stock solution of Fritillaria sulfonate and a methanol solution are added successively. After ultrasonic extraction, the extract is filtered through a membrane to obtain a filtrate;

[0011] S2, the filtrate is subjected to ultra-high performance liquid chromatography-evaporative light scattering detection, that is, the content of Fritillaria sulfonate in the Fritillaria to be tested is determined;

[0012] The chromatographic conditions of the ultra-high performance liquid chromatography-evaporative light scattering detection are as follows:

[0013] The mobile phase was ultrapure water and acetonitrile in a volume ratio of 85:15;

[0014] The elution mode is isocratic elution;

[0015] The chromatographic column is ACQUITY HSS T3 chromatographic column, specifications: 1.8μm, 2.1×100mm;

[0016] The flow rate is 0.2-0.4 mL / min, preferably 0.2 mL / min;

[0017] The column temperature is 25°C-35°C, preferably 30°C.

[0018] In the quantitative analysis method of the present invention, the evaporative light scattering detection conditions of the ultra-high performance liquid chromatography-evaporative light scattering detection are as follows:

[0019] The drift tube temperature was 65°C, the nebulizer temperature was 45°C, and the gas flow rate was 1.1 mL / min;

[0020] The experiment found that the drift tube temperature has a great influence on the response value (peak area) of the target detection, showing a trend that the response value first increases and then decreases with the increase of the drift tube temperature. When the drift tube temperature is 65℃, the response value of the target is the largest and the chromatographic peak shape is the best, so the drift tube temperature is selected to be 65℃.

[0021] The experiment found that the nebulizer temperature has a great influence on the response value of the target object detection, showing a trend of increasing first and then decreasing with the increase of the nebulizer temperature. When the drift tube temperature is 45℃, the response value of the target object is the largest and the chromatographic peak shape is the best, so the nebulizer temperature is selected to be 45℃.

[0022] Experiments have shown that the gas flow rate has a significant effect on the response value of target detection, showing a trend of first increasing and then decreasing with the increase of gas flow rate. When the gas flow rate is 1.1 ml / min, the response value of the target is the largest and the chromatographic peak shape is the best, so the gas flow rate is determined to be 1.1 ml / min.

[0023] In the quantitative analysis method of the present invention, the concentration of the reference substance stock solution is 0.1-0.4 mg / mL.

[0024] In the quantitative analysis method of the present invention, the concentration of the methanol solution is 60%. Experiments have found that the extraction solvent has a greater influence on the extraction effect of Fritillaria spicatrum. When 40% methanol and 80% methanol are used for extraction, there is a matrix enhancement effect, and the extraction rate of 60% methanol is 98.84%, so 60% methanol is selected as the extraction solvent.

[0025] In the quantitative analysis method of the present invention, the ultrasonic extraction time is 60 minutes. Experiments have shown that the recovery rates of ultrasonic extraction for 60 and 75 minutes are relatively high, and the recovery rates of the two are relatively similar. Under the condition of ensuring a high recovery rate, 60 minutes is selected as the ultrasonic extraction time for the sake of saving time.

[0026] In the quantitative analysis method of the present invention, the usage ratio of the tested Fritillaria to the reference substance stock solution is 1g:0.2-0.6ml, and the usage ratio of the tested Fritillaria to the methanol solution is 1g:10-20ml.

[0027] The present invention has the following beneficial technical effects:

[0028] The present invention is based on UPLC-ELSD technology, and establishes a qualitative and quantitative detection method suitable for sulfur-fumigated Fritillaria-type Chinese medicinal materials. The method has the advantages of low cost, simple operation, high sensitivity, rapidity, and a wide range of applications. It has important practical application value and solves the problem of the narrow application range of existing detection methods to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 are chromatograms of different samples.

[0030] Figure 2 It is the linear relationship of the detection method of the present invention. DETAILED DESCRIPTION

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Instruments and reagents used in the following examples: UPLC high performance liquid chromatograph (Agilent Technologies, Inc., model 1290Ⅱ); 1 / 10,000 analytical balance (Mettler-Toledo (Shanghai) Co., Ltd., model EL104); acetonitrile (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographically pure, batch number: 20230814), methanol (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographically pure, batch number: 20230714); water is Wahaha purified water; and the sulfonic acid fritillariae succinate reference substance is homemade.

[0033] Embodiment 1,

[0034] 1. Preparation of reference solution

[0035] Accurately weigh 10.57 mg of Fritillaria octyldodecene reference substance, place it in a 10 ml volumetric flask, add 70% methanol to dilute to the mark, and obtain a 1.057 mg / mL reference substance stock solution. Accurately pipette 1 ml of the reference substance stock solution, place it in a 5 ml volumetric flask, and dilute to the mark with 70% methanol to obtain a 0.2114 mg / mL reference substance solution.

[0036] 2. Preparation of positive samples

[0037] Take about 1.0g of medicinal powder (pass 60 mesh), weigh it accurately, put it in a 50mL centrifuge tube, add 10mL of 60% methanol accurately, and extract by ultrasonic (power 120W, frequency 40kHZ) for 60min. Take it out and let it cool, then weigh it. Pass the extract through a 0.22μm filter membrane to obtain the test solution.

[0038] 3. Chromatographic conditions

[0039] Ultrapure water (A)-acetonitrile (B) (85:15) was used as the mobile phase, and the elution was isocratic; ACQUITY HSS T3 (Waters, USA, 1.8 μm, 2.1×100 mm) was used as the chromatographic column; flow rate was 0.2 mL / min; column temperature was 30°C; ELSD detector parameters: drift tube temperature was 65°C, nebulizer temperature was 45°C, and gas flow rate was 1.1 mL / min.

[0040] The chromatograms of the control, positive sample and reagent blank (60% methanol in water) are as follows: Figure 1 As shown, it can be seen that the reference substance and the positive sample have the same chromatographic peak at the same retention time (about 5 minutes), and the solvent blank has no interference with the detection of the target component.

[0041] Embodiment 2, the extraction condition of Fritillaria medicinal material

[0042] 1. Extraction time

[0043] Take 5 portions of blank Fritillaria cirrhosa powder, each portion is about 1.0g (passed through 60 mesh), accurately weighed, placed in a 50ml centrifuge tube, accurately added with 0.5ml of Fritillaria sulphate reference stock solution, and then accurately added with 10mL of 70% methanol, respectively, ultrasonically extracted (power 120W, frequency 40kHZ) for 30, 45, 60, 75min, then weighed, the extract passed through a 0.22μm filter membrane, detected according to the chromatographic conditions in Example 1, and calculated the recovery rate.

[0044] From Table 2, the recovery rates of ultrasonic extraction for 60 and 75 min are relatively high, and the recovery rates of the two are relatively close. Under the premise of ensuring a high recovery rate, 60 min is selected as the ultrasonic extraction time for the sake of saving time.

[0045] Table 1 Extraction effect of different ultrasonic extraction times

[0046]

[0047]

[0048] 2. Extraction solvent

[0049] Take 6 portions of blank Fritillaria cirrhosa powder, each portion is about 1.0g (passed through 60 mesh), accurately weighed, placed in a 50ml centrifuge tube, accurately added with 0.5ml of Fritillaria sulphate reference stock solution, and then accurately added with 10mL of methanol of different concentrations (40%, 60%, 80%), ultrasonic extraction (power 120W, frequency 40kHZ) for 60min, make up the weight, the extract is filtered through a 0.22μm filter membrane, and detected according to the chromatographic conditions in Example 1, and the recovery rate is calculated.

[0050] From Table 2, we can see that the extraction solvent has a great influence on the extraction effect of Fritillaria spicatrum. Both 40% methanol and 80% methanol extraction have matrix enhancement effect. The extraction efficiency of 60% methanol is 98.84%, which meets the requirements, so 60% methanol is selected as the extraction solvent.

[0051] Table 2 Extraction effects of different extraction solvents

[0052] Extraction solvent Sample weight / g Addition amount / ug Measured amount / ug Recovery rate / % 40% methanol 1.0001 528.5 732.034 137.57 60% methanol 1.0002 528.5 536.137 98.84 80% Methanol 1.0001 528.5 665.589 120.01

[0053] Example 3, ELSD detection conditions of Fritillaria medicinal materials

[0054] 1. Drift tube temperature

[0055] Under the conditions of constant atomizer temperature (40°C) and gas flow rate (1.0ml / min), the drift tube temperature of ELSD detection was investigated. From Table 3, the drift tube temperature has a great influence on the response value (peak area) of the target detection, showing a trend that the response value first increases and then decreases with the increase of the drift tube temperature. When the drift tube temperature is 65°C, the response value of the target is the largest and the chromatographic peak shape is the best, so the drift tube temperature is selected to be 65°C.

[0056] Table 3 Response values ​​of target detection at different drift tube temperatures

[0057] Temperature / ℃ Peak area / A 45 612.0 50 651.0 55 654.5 60 676.9 65 748.9 70 694.3

[0058] 2. Atomizer temperature

[0059] Under the conditions of fixed drift tube temperature (65°C) and gas flow rate (1.0ml / min), the nebulizer temperature was investigated. From Table 4, the nebulizer temperature has a great influence on the response value of target detection, showing a trend of increasing and then decreasing with the increase of nebulizer temperature. When the drift tube temperature is 45°C, the response value of the target is the largest and the chromatographic peak shape is the best, so the nebulizer temperature is selected to be 45°C.

[0060] Table 4 Response values ​​of target detection at different atomizer temperatures

[0061] Temperature / ℃ Peak area / A 40 748.9 43 704.0 45 775.1 47 718.6 50 737.4

[0062] 3. Gas flow

[0063] Under the conditions of fixed drift tube temperature (65°C) and nebulizer temperature (45°C), the gas flow rate was investigated. From Table 5, the gas flow rate has a great influence on the response value of target detection, showing a trend of increasing first and then decreasing with the increase of gas flow rate. When the gas flow rate is 1.1ml / min, the response value of the target is the largest and the chromatographic peak shape is the best, so the gas flow rate is determined to be 1.1ml / min.

[0064] Table 5 Gas flow rate investigation results

[0065] Gas flow / ml / min Peak area / A 1.0 717.9 1.1 867.7 1.2 776.1

[0066] Example 4: Methodological Investigation

[0067] 1. Linear relationship

[0068] 1.5, 2, 2.5, 3, 4 μl of the reference solution (0.2114 mg / ml) in Example 1 was accurately drawn and measured according to the chromatographic conditions in Example 1, and the peak area was recorded (Table 6). With the injection volume (μg) as the abscissa and the peak area as the ordinate, a linear regression was performed to obtain the regression equation: Y = 1003.9693x-195.1514, and the correlation coefficient (r 2 ) is 0.9999, indicating that the linear relationship of Fritillaria succinate is good in the range of 0.3171ug~0.8456ug.

[0069] Table 6 Linear relationship investigation results

[0070] serial number Concentration / ug Peak area / A 1 0.3171 124.4 2 0.4228 224.7 3 0.5285 331.7 4 0.6342 438.9 5 0.8456 650.6

[0071] 2. Precision test

[0072] Take the reference solution, inject it 6 times continuously according to the chromatographic conditions in Example 1, record the peak area value of Fritillaria octyl sulfur, and calculate its RSD value. As shown in Table 7, the average peak area of ​​6 consecutive injections is 231.81, and the RSD value is 2.58%, indicating that the instrument has good precision.

[0073] Table 7 Precision investigation results

[0074] Serial number Peak area Mean / A RSD / % 1 225.4 2 240.0 3 231.8 231.81 2.58 4 233.6 5 235.6 6 224.5

[0075] 3. Repeatability test

[0076] Accurately weigh 6 portions of the same Fritillaria cirrhosa sample, each about 1 g, and accurately add 528.5 ug of Fritillaria sulphate to prepare the test solution according to the optimal method determined in Example 2, and detect according to the chromatographic conditions in Example 1, and calculate the RSD value of the recovery rate of Fritillaria sulphate. As shown in Table 8, the recovery rate of the tested samples is 97.60% to 100.13%, and the RSD value is 1.03%, indicating that the method established by the present invention has good repeatability.

[0077] Table 8 Repeatability test results

[0078] serial number Addition amount / ug Measured amount / ug Recovery rate / % RSD / % 1 528.5 529.2 100.13 2 528.5 515.8 97.60 3 528.5 518.2 98.06 1.03 4 528.5 526.2 99.56 5 528.5 527.9 99.89 6 528.5 523.2 99.00

[0079] 4. Stability test

[0080] Take 1 blank sample of Fritillaria cirrhosa, accurately add 528.5ug of Fritillaria sulphate, prepare the test solution according to the optimal method determined in Example 2, place at room temperature for 0, 2, 4, 6, 12, and 24 hours, and measure according to the chromatographic conditions in Example 1 to calculate the recovery rate and RSD value of Fritillaria sulphate. As shown in Table 9, the recovery rate of the tested samples is 103.45% to 105.55%, and the RSD value is 1.03%, indicating that the test solution has good stability within 24 hours.

[0081] Table 9 Repeatability test results

[0082] Time / h Addition amount / ug Measured amount / ug Recovery rate / % RSD / % 0 528.5 0.5180 98.02 2 528.5 0.5295 100.20 4 528.5 0.5312 100.50 1.09 6 528.5 0.5204 98.47 12 528.5 0.5305 100.38 24 528.5 0.5221 98.80

[0083] 5. Recovery rate test

[0084] Accurately weigh about 1.0g of blank flat Fritillaria sample, and accurately add low (200μL), medium (400μL), and high (600μL) concentrations of Fritillaria sulphate reference stock solution (1.057mg / mL), respectively, and each concentration is in triplicate, for a total of 9 parts. Prepare the test solution according to the optimal method determined in Example 2, detect according to the chromatographic conditions in Example 1, and calculate the recovery rate and RSD value of Fritillaria sulphate. As can be seen from Table 10, the recovery rate of the tested samples is 97.83% to 104.86%, the RSD value is 2.39%, and the sample recovery rate meets the requirements of the 2020 edition of the "Chinese Pharmacopoeia", indicating that the method established by the present invention has good accuracy.

[0085] Table 10 Sample recovery test results

[0086] serial number Sample weight / g Addition volume / μL Addition amount / ug Measured amount / ug Recovery rate / % D-1 1.0001 200 221.4 208.5 98.63 D-2 1.0002 200 221.4 213.2 100.86 D-3 1.0003 200 221.4 206.8 97.83 Z-1 1.0002 400 422.8 417.0 98.63 Z-2 1.0002 400 422.8 430.8 101.90 Z-3 1.0000 400 422.8 421.0 99.58 G-1 1.0001 600 634.2 657.2 103.63 G-2 1.0002 600 634.2 665.0 104.86 G-3 1.0002 600 634.2 645.8 101.83 RSD / % 2.39

[0087] Example 5: Sample detection

[0088] Take each medicinal material powder, prepare the test solution according to the optimal solution determined in Example 2, and detect according to the chromatographic conditions in Example 1. The content of Fritillaria succinic acid (ug / g) in the tested samples is shown in Table 11.

[0089] Table 11 Sample test results

[0090] Sample source Content / ug / g Flat Fritillary 1 -- Flat Fritillary 2 -- Flat Fritillary 3 65.45 Flat Fritillary 4 9.58 Fritillaria 1 38.45 Fritillaria 2 75.64 Fritillaria 3 8.65 Hubei Fritillaria 1 75.02 Hubei Fritillaria 2 338.44 Hubei Fritillaria 3 659.18 Hubei Fritillaria 4 8.10 Fritillaria thunbergii 1 96.49 Fritillaria thunbergii 2 82.54 Fritillaria thunbergii 3 142.58 Fritillaria 1 36.25 Fritillaria 2 75.68 Fritillaria 3 26.67

[0091] Note: “--” means not detected

[0092] From the data in Table 11, it can be seen that the sulfur fumigation marker Fritillaria sulphur was detected in 2 batches of Fritillaria balsamifera samples, 3 batches of Fritillaria ivorita, 4 batches of Fritillaria hupehensis, 3 batches of Fritillaria thunbergii, and 3 batches of Fritillaria chuanxiong, with a content of 9.58-659.18ug / g. In terms of content, the sulfur fumigation degree of each batch of medicinal materials is different. The sulfur fumigation of the two batches of Hubei Fritillaria medicinal materials is more serious, and the content of Fritillaria sulphur is 338.44ug / g and 659.18ug / g respectively. The quantitative detection of the sulfur fumigation marker Fritillaria sulphur in Fritillaria medicinal materials can reflect whether the medicinal materials are sulfur fumigated and the degree of sulfur fumigation to a certain extent, which is of great significance for market supervision.

Claims

1. A quantitative analysis method for sulfur-fumigation marker sulfonate fritillariae in fritillariae Chinese medicinal materials, comprising the following steps: S1. The tested Fritillaria is crushed and sieved, and a reference substance stock solution of Fritillaria sulfonate and a methanol solution are added successively. After ultrasonic extraction, the extract is filtered through a membrane to obtain a filtrate; S2, the filtrate is subjected to ultra-high performance liquid chromatography-evaporative light scattering detection, that is, the content of Fritillaria sulfonate in the Fritillaria to be tested is determined; The chromatographic conditions of the ultra-high performance liquid chromatography-evaporative light scattering detection are as follows: The mobile phase was ultrapure water and acetonitrile in a volume ratio of 85:15; The elution mode is isocratic elution; The chromatographic column is ACQUITY HSS T3 column; Flow rate 0.2-0.4 mL / min; The column temperature is 25℃-35℃.

2. The quantitative analysis method according to claim 1, characterized in that: The evaporative light scattering detection conditions of the ultra-high performance liquid chromatography-evaporative light scattering detection are as follows: The drift tube temperature was 65 °C, the nebulizer temperature was 45 °C, and the gas flow rate was 1.1 mL / min.

3. The quantitative analysis method according to claim 1 or 2, characterized in that: The concentration of the reference substance stock solution is 0.1-0.4 mg / mL.

4. The quantitative analysis method according to claim 1 or 2, characterized in that: The concentration of the methanol solution is 60%.

5. The quantitative analysis method according to claim 1 or 2, characterized in that: The ultrasonic extraction time is 60 min.

6. The quantitative analysis method according to claim 1 or 2, characterized in that: The dosage ratio of the tested Fritillaria to the reference substance stock solution is 1 g: 0.2-0.6 ml; The dosage ratio of the tested Fritillaria to the methanol solution is 1g:10-20ml.

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