Radix stemonae medicinal material and method for checking adulteration of asparagus in loquat cough-relieving preparation containing radix stemonae raw material

Abstract

The invention discloses an adulteration inspection method for asparagus in a radix stemonae medicinal material and a loquat cough-relieving preparation containing the radix stemonae raw material. The adulteration inspection method comprises the following steps: (1) carrying out chromatography, mass spectrum condition and system applicability test; (2) preparing a reference solution; (3) preparing a test solution; according to the method, original neodioscin is taken as a detection index, a high performance liquid chromatography-mass spectrometry method is adopted for detection, the operation is simple, the specificity is high, whether the radix stemonae medicinal material or the loquat cough-relieving preparation containing the radix stemonae raw material has the phenomenon of adulteration of asparagus or not can be rapidly and accurately detected by adopting the method, and the quality of the loquat cough-relieving preparation containing the radix stemonae raw material can be ensured. Furthermore, the quality, safety and effectiveness of the radix stemonae medicinal material and the loquat cough-relieving preparation are ensured. The method provided by the invention can provide technical support for feeding supervision of the radix stemonae medicinal material and the loquat cough-relieving preparation containing the radix stemonae raw material.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for detecting adulteration of Asparagus cochinchinensis in a Radix Stemonae medicinal material and a loquat cough-relieving preparation containing the Radix Stemonae raw material. Background Art

[0002] Stemona radix (Stemona sessilifolia) (Miq.) Miq., Stemona japonica (Bl.) Miq., or Stemona tuberosa Lour. (Stemona sessilifolia) (Miq.), Stemona japonica (Bl.) Miq., or Stemona tuberosa Lour., all members of the Stemonaceae family. It has a sweet, bitter, and slightly warming nature and enters the Lung meridian. It moistens the lungs, lowers qi, relieves coughs, and kills insects and lice. It is commonly used for new and chronic coughs, coughs caused by tuberculosis, and sudden coughs. It is also used externally for head and body lice, pinworms, and vaginal itching. Honeyed Stemona radix moistens the lungs and relieves coughs, and is used for coughs caused by yin deficiency and fatigue. Stemona radix contains multiple active ingredients, including alkaloids such as stemonine, protostemonine, and parastemonine, as well as proteins and lipids. These components work together to relieve cough and asthma, and expel parasites. It has a certain inhibitory effect on tuberculosis, diphtheria, and Staphylococcus aureus, and has been used as an adjunct treatment for tuberculosis.

[0003] Asparagus Asparagus 0fficinalis L. Asparagus is an upright herbaceous plant of the genus Asparagus in the family Liliaceae. It can reach up to 1 meter in height; its stems are smooth; its leaves are nearly flattened cylindrical with slightly blunt edges, and its scale-like leaves have short or nearly no spurs at the base. Its flowers are axillary, greenish-yellow, with smaller female flowers; its berries are red when ripe; its flowering period is May to June, and its fruiting period is September to October. Asparagus is called "Asparagus" because it resembles a gypsophila when it emerges from the ground, and its stems and branches grow leaves similar to those of a cypress. Asparagus feeds on young stems. Young stems harvested before emerging from the ground are white and tender, and are called white asparagus; young stems exposed to light after emerging from the ground are called green asparagus. Asparagus is a common vegetable, and its rhizomes are readily available and inexpensive. The dried tubers of Stemona radiata are similar in shape and color to those of Asparagus, making them difficult to distinguish, especially after slicing or processing. At present, there is a phenomenon in the market of using Asparagus cochinchinensis mixed with Stemona radix, but it is difficult to identify it using traditional identification methods. The addition of Asparagus cochinchinensis into Stemona radix will seriously affect the quality, efficacy and safety of Stemona radix.

[0004] Loquat cough-relieving preparation is a compound preparation composed of seven herbs, including loquat leaves, stemona, mulberry bark, scutellaria baicalensis, menthol, and platycodon. The loquat cough-relieving preparations included in the 2020 edition of the Pharmacopoeia of the People's Republic of China are loquat cough-relieving granules, loquat cough-relieving capsules, and loquat cough-relieving soft capsules, including granules, capsules, and soft capsules. These preparations have the effect of relieving cough and reducing phlegm, and are clinically used for cough and sputum caused by phlegm-heat in the lungs; and for bronchitis with the above symptoms. Stemona is one of the important raw materials of loquat cough-relieving preparations. If Asparagus cochinchinensis is added to the raw materials used in loquat cough-relieving preparations, it will seriously affect the efficacy and drug safety of loquat cough-relieving preparations. Therefore, developing a simple, fast and accurate method for detecting adulteration of Asparagus cochinchinensis in Stemona radix and loquat cough suppressant preparations containing Stemona radix is ​​of great significance for ensuring the quality of Stemona radix and loquat cough suppressant preparations containing Stemona radix asparagus and their safety and effectiveness in clinical use. Summary of the Invention

[0005] The present invention aims to provide a method for detecting adulteration of Asparagus cochinchinensis in Stemona radix and loquat cough-relieving preparations containing Stemona radix. This method uses protoneodioscin as a detection indicator and employs high-performance liquid chromatography-mass spectrometry. This method can quickly and accurately detect the presence of Asparagus cochinchinensis in Stemona radix or loquat cough-relieving preparations containing Stemona radix, thereby ensuring the quality, safety, and effectiveness of the Stemona radix and loquat cough-relieving preparations.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for detecting adulteration of Asparagus cochinchinensis in a Stemona herb and a loquat cough-relieving preparation containing the Stemona herb, wherein the loquat cough-relieving preparation includes loquat cough-relieving granules, loquat cough-relieving capsules, and loquat cough-relieving soft capsules. The method comprises the following steps: (1) Chromatographic, mass spectrometric conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (chromatographic column inner diameter 2.1 mm); acetonitrile was used as mobile phase A, 0.1% formic acid 10 mmol / L ammonium formate solution was used as mobile phase B, and gradient elution was performed as specified in the table below; the flow rate was 0.25 ml / min; the column temperature was 35 °C; the mass spectrometer was used in electrospray positive ionization mode (ESI + ), multiple reaction monitoring (MRM); select mass-to-charge ratio m / z 1031.5→869.3 and m / z 1031.5→577.3 is used as the detection ion pair for original dioscin; 5 μl of the reference solution is injected, and the signal-to-noise ratio of the MRM chromatographic peak measured by the above detection ion pair should be greater than 10:1;

[0007] (2) Preparation of reference solution Reference solution of Radix Stemonae Radix: Take an appropriate amount of original new dioscin protoneodioscin reference substance, accurately weigh it, place it in a 50ml volumetric flask, add 70% ethanol to dissolve it and dilute it to the scale, shake it well, and then dilute it with 70% ethanol to a solution containing 46ng per 1ml. Reference solution of loquat cough suppressant preparation: Take an appropriate amount of original new dioscin protoneodioscin reference substance, accurately weigh it, place it in a 50ml volumetric flask, add 70% ethanol to dissolve it and dilute it to the scale, shake it well, and then dilute it with 70% ethanol to a solution containing 35ng per 1ml. (3) Preparation of test solution Stemona tuberosa test solution: grind the product, pass through a No. 3 sieve, mix well, take 0.2 g, accurately weigh, place in a stoppered conical flask, accurately add 50 ml of 70% ethanol, weigh the weight, ultrasonicate for 40 minutes, let cool, weigh again, make up the lost weight with 70% ethanol, shake well, and filter through a 0.22 μm filter membrane. Loquat cough suppressant test solution: For granules, take the product listed in the dosage difference, grind it into powder, and take approximately 3 g. Weigh accurately. For capsules and soft capsules, take the product listed in the dosage difference, mix well, and take an amount equivalent to 2 capsules. Weigh accurately, place in a stoppered conical flask, and accurately add 50 ml of 70% ethanol. Weigh accurately, sonicate for 40-45 minutes, cool, and weigh again. Make up the loss with 70% ethanol. Shake well, filter, and collect the filtrate. (4) Determination: Accurately pipette 1-5 μl of the reference solution and the test solution, inject them into the high performance liquid chromatography-mass spectrometer, measure, and record the chromatogram; (5) Judgment principle: ① If the chromatographic peak corresponding to the chromatographic peak of the reference solution does not appear in the extracted ion current chromatogram of the test sample, it is considered as not detected; ② If the chromatographic peak corresponding to the chromatographic peak of the reference solution appears in the extracted ion current chromatogram of the test sample, and the chromatographic peak of the reference solution appears in the extracted ion current chromatogram of the test sample, m / z If the chromatographic peak area value of 1031.5→869.3 is not greater than the corresponding peak area value in the reference solution, it is considered as not detected; ③ In the extracted ion current chromatogram of the test sample, the chromatographic peak corresponding to the reference solution chromatogram appears at the same time, and the chromatographic peak of the test sample is m / z If the chromatographic peak area value of 1031.5→869.3 is greater than the corresponding peak area value in the reference solution, it is considered detected; (6) Result determination: In the extracted ion current chromatogram of the test sample, no chromatographic peak corresponding to the chromatogram of the reference solution should be detected.

[0008] Furthermore, in the method for checking adulteration of Asparagus cochinchinensis in the above-mentioned Radix Stemonae medicinal materials and loquat cough-relieving preparations containing Radix Stemonae raw materials, in step (3) of preparing the test solution, the ultrasonic treatment has an ultrasonic power of 560 W and a frequency of 45 kHz.

[0009] The beneficial effects of the present invention are: The inventors of this application determined the content of protoneodioscin in Asparagus japonicus and Stemona radix. The results showed that the content of protoneodioscin in Asparagus japonicus was high, while protoneodioscin was not detected in Stemona radix. Therefore, the present invention established a method for detecting adulteration of Asparagus japonicus in Stemona radix and loquat cough-relieving preparations containing Stemona radix using high-performance liquid chromatography-mass spectrometry, using protoneodioscin as a detection indicator. This adulteration detection method is simple and easy to perform, with good precision, repeatability, and stability, and strong specificity. Other medicinal ingredients in loquat cough-relieving granules, capsules, and soft capsules do not interfere with the determination, and the determination results are accurate, stable, and reliable. This method can quickly and accurately detect the presence of adulterated Asparagus cochinchinensis in Stemona tuber or loquat cough-relieving preparations containing Stemona tuber, investigate the feeding status of Stemona tuber in samples, and thereby ensure the quality of Stemona tuber and loquat cough-relieving preparations, as well as their safety and effectiveness in clinical use. The adulteration detection method provided by this invention can provide technical support for the feeding supervision of Stemona tuber and loquat cough-relieving preparations containing Stemona tuber. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 TIC / MRM diagram of the original new dioscin protoneodioscin reference substance; Figure 2 Negative TIC / MRM chart of Loquat Cough Granules lacking Stemona tuberosa; Figure 3 TIC / MRM graph of the negative sample of Loquat Cough Granules (supplied by D Co., Ltd., batch number: UB30003, no original dioscin was detected); Figure 4 TIC / MRM diagram of the positive sample of Loquat Cough Granule (supplied by Company A, batch number: 23030901, original dioscin detected); Figure 5 Loquat cough capsule lacks the negative TIC / MRM chart of Stemona radix; Figure 6 TIC / MRM diagram of the negative sample of Loquat Cough Capsule (supplied by Company B, batch number: S240112, no original dioscin was detected); Figure 7 TIC / MRM diagram of the positive sample of Loquat Cough Capsule (supplied by Company B, batch number: S240411, original dioscin was detected); Figure 8 The TIC / MRM diagram of loquat cough-relieving soft capsule lacks the negative TIC / MRM diagram of Stemona radix; Figure 9 TIC / MRM diagram of the positive sample of Loquat Cough Soft Capsule (supplied by Company C, batch number: 20240101); Figure 10 TIC / MRM diagram of the reference herbal medicine Stemona tuberosa (batch number: 121221-201604, purchased from China National Institute for Food and Drug Control); Figure 11 TIC / MRM chart of the control herbal medicine Stemona erecta (batch number: 121588-201502, purchased from the China National Institute for Food and Drug Control); Figure 12 TIC / MRM diagram of Asparagus cochinchinensis medicinal material; Figure 13 Standard curve of original new dioscin; Figure 14 Detection limit diagram of the original new dioscin; Figure 15 TIC / MRM diagram of Loquat Cough Granules (supplied by Company A, batch number: 23030901). DETAILED DESCRIPTION Example 1

[0011] Adulteration inspection method for Asparagus cochinchinensis in Radix Stemonae and loquat cough suppressant preparations containing Radix Stemonae 1. Instruments and test drugs Instruments: Agilent 1290 Ultra-High Performance Liquid Chromatography System; Agilent 6490 Triple Quadrupole MS System; ML204 Electronic Analytical Balance (Mettler-Toledo Instruments Shanghai Co., Ltd.). Reagents: Acetonitrile was chromatographic grade; water was ultrapure. All other reagents were of analytical grade. Reference Standard: Protoneodioscin Reference Standard (purchased from Shanghai Shidande Standard Technology Service Co., Ltd., for content determination, batch number TS24C091-DT02, content: 100%).

[0012] Medicinal Materials: 15 batches of Stemona tuberosa were sourced from Baise, Guangxi Zhuang Autonomous Region; Funing, Guangnan, and Wenshan Counties, Yunnan Province; Xingyi and Bijie, Guizhou Province. 29 batches of Asparagus cochinchinensis were sourced from Nanning, Guangxi Zhuang Autonomous Region; Cao County, Shandong Province; and Heze. See Table 1 for details. 175 batches of loquat cough suppressant preparations (including granules and capsules) were samples for national random inspection in 2024.

[0013]

[0014]

[0015] 2. Experimental Conditions Investigation and Method Verification 2.1 Chromatographic, mass spectrometric conditions and system suitability test The chromatographic column was MicroPulite XP tC18, 1.7 μm, 2.1 × 100 mm, at a column temperature of 35°C and a flow rate of 0.25 ml / min. Experiments were conducted to compare three mobile phase systems: acetonitrile-water, acetonitrile-0.1% formic acid, and acetonitrile-0.1% formic acid 10 mmol / L ammonium formate solution. The results showed that when acetonitrile-0.1% formic acid 10 mmol / L ammonium formate solution was used as the mobile phase and the ionization mode was positive, the protoneodioscin (C 51 H 84 O 22 ) had a larger response, so the chromatographic conditions were determined as follows: acetonitrile as mobile phase A, 0.1% formic acid 10mmol / L ammonium formate solution as mobile phase B, gradient elution program as shown in Table 2; flow rate of 0.25ml / min; column temperature of 35℃; injection volume of 2μl.

[0016]

[0017] Mass spectrometry conditions: The mass spectrometry conditions were optimized with the reference solution of Protoneodioscin (10 μg / ml), and the scans were performed in positive and negative ion modes. It was found that when the ionization mode was positive, Protoneodioscin (C 51 H 84 O 22 ) had a large response, with the parent ion at m / z 1031.5. The optimized parameters are detailed in Table 3. The mass spectrometry conditions were as follows: electrospray ionization (ESI) in positive ion mode with MRM detection; drying gas temperature: 200°C; drying gas flow rate: 12 L / min; nebulizer gas pressure: 20 psi; sheath gas temperature: 350°C; sheath gas flow rate: 10 L / min; capillary voltage: 4.0 kV; and nozzle voltage: 500 V.

[0018]

[0019] System suitability: Take 5μl of the original new dioscin protoneodioscin reference solution and inject it. The signal-to-noise ratio of the MRM chromatographic peak determined by the above ion pair determination should be greater than 10:1.

[0020] 2.2 Investigation of test solution preparation The granules were sampled from Company A, batch number 23030901; the capsules were sampled from Company B, batch number S230817; and the soft capsules were sampled from Company C, batch number 20240101.

[0021] Since the original dioscin was not detected in the capsule test sample (batch number S230817), 100μl of original dioscin solution (10.016μg / ml) was precisely added after sampling, and the samples were dried before conducting the experiment.

[0022] The medicinal materials of Stemona tuberosa (sample No. 1 in Table 1) and Asparagus cochinchinensis (sample No. 16 in Table 1) were taken as the research objects. The medicinal materials of Asparagus cochinchinensis were crushed, passed through a No. 3 sieve, and mixed.

[0023] 2.2.1 Selection of extraction method Ultrasonic extraction and heating reflux were compared respectively.

[0024] Ultrasonic extraction: Take an appropriate amount of granules, grind them into powder, take about 3g, and weigh accurately; take an appropriate amount of the contents of the soft capsule, the amount equivalent to 2 capsules (1.1g); take 0.2g of Asparagus cochinchinensis medicinal material, weigh accurately, place it in a stoppered conical flask, accurately add 50ml of water, weigh the weight, and ultrasonically treat for 40 minutes (power 560W, frequency 45kHz), let cool, weigh again, make up for the lost weight, shake well, and filter through a 0.22μm filter membrane.

[0025] Take an appropriate amount of the contents of the capsule, the amount equivalent to 2 capsules (0.5g), accurately weigh, place in a stoppered conical flask, accurately add 100μl of the original dioscin solution (10.08μg / ml), dry, then accurately add 50ml of water, weigh, ultrasonically treat for 40 minutes (power 560W, frequency 45kHz), let cool, weigh again, make up for the lost weight, shake well, and filter through a 0.22μm filter membrane.

[0026] Heating and reflux: Take an appropriate amount of granules, grind them into powder, take about 3g, and weigh accurately; take an appropriate amount of the contents of the soft capsule, the amount equivalent to 2 capsules (1.1g); take 0.2g of Asparagus cochinchinensis medicinal material, weigh accurately, place it in a stoppered conical flask, accurately add 50ml of water, weigh the weight, heat and reflux for 40 minutes, cool, weigh again, make up the lost weight with water, shake well, and filter through a 0.22μm filter membrane to obtain.

[0027] Take an appropriate amount of the contents of the capsule, which is equivalent to 2 capsules (0.5g), and take 0.2g of the powder of Stemona tuberosa, accurately weigh it, and place it in a stoppered conical flask. Accurately add 100μl of the original dioscin solution (10.016μg / ml), dry it, and then accurately add 50ml of water, weigh it, heat it under reflux for 40 minutes, cool it, weigh it again, make up the lost weight with water, shake it well, and filter it through a 0.22μm filter membrane.

[0028] The results showed that the contents of the tested components by ultrasonic extraction and heating reflux extraction were consistent. The ultrasonic extraction method was used for extraction, as shown in Table 4.

[0029]

[0030] 2.2.2 Selection of extraction solvent The extraction effects of five solvents, including water, 30% ethanol, 70% methanol, 70% ethanol and ethanol, were investigated respectively.

[0031] Take an appropriate amount of granules, grind them into powder, take about 3g, and weigh accurately; take an appropriate amount of the contents of the soft capsule, the amount equivalent to 2 capsules (1.1g); take 0.2g of Asparagus cochinchinensis medicinal material, weigh accurately, and place it in a stoppered conical flask, take a total of 10 portions, and make two portions into a group. To each group, accurately add water, 30% ethanol, 70% methanol, 70% ethanol, and ethanol, respectively, and extract and determine the content of the target component according to the proposed method.

[0032] Take an appropriate amount of the contents of the capsule, the amount equivalent to 2 capsules (0.5g), take 0.2g of the powder of Stemona tuberosa, accurately weigh it, put it in a stoppered conical flask, accurately add 100μl of the original dioscin solution (10.016μg / ml), dry it, take a total of 10 portions, and make two portions into a group. To each group, accurately add water, 30% ethanol, 70% methanol and 70% ethanol, ethanol, respectively. Extract and determine the content of the target component according to the proposed method.

[0033] The test results showed that the content of the tested components was the highest when extracted with 70% ethanol, so 70% ethanol was selected as the extraction solvent. See Table 5 for details.

[0034]

[0035] 2.2.3 Selection of extraction solvent dosage The extraction effects of different 70% ethanol dosages (25, 50, 100 ml) were investigated. The results showed that the content of loquat cough capsules and soft capsules was low when 70% ethanol dosage (25) was used, while the content of the tested components was basically the same when 70% ethanol dosage (50, 100 ml) was used. Therefore, 50 ml of 70% ethanol was selected. See Table 6 for details.

[0036]

[0037] 2.2.4 Selection of extraction time The effects of ultrasonic extraction for 30 minutes, 45 minutes, and 60 minutes were compared.

[0038] Take an appropriate amount of granules, grind them into powder, take about 3g, and weigh accurately; take an appropriate amount of the contents of the soft capsule, the amount equivalent to 2 capsules (1.1g); take 0.2g of Asparagus cochinchinensis medicinal materials, weigh accurately, and place in a stoppered conical flask, take a total of 6 portions, two portions as a group, and ultrasonically extract each group for 30 minutes, 45 minutes, and 60 minutes respectively, and prepare according to the proposed method.

[0039] Take an appropriate amount of the contents of the capsule, the amount equivalent to 2 capsules (0.5g), accurately weigh, place in a stoppered conical flask, accurately add 100μl of the original dioscin solution (10.016μg / ml), dry, take a total of 6 portions, two portions as a group, each group is ultrasonically extracted for 30 minutes, 45 minutes, and 60 minutes respectively, and prepared according to the proposed method.

[0040] The test results showed that the content of the tested components was not much different. The extraction time was 45 minutes. See Table 7 for details.

[0041]

[0042] Based on the above research, the preparation method of the test solution is as follows: Loquat cough granules and capsules: Take the product under the item of difference in filling quantity, grind the granules into fine powder, and take about 3g. For capsules and soft capsules, take the contents under the item of difference in filling quantity, mix them evenly, and take the amount equivalent to 2 capsules (0.5g for capsules, 1.1g for soft capsules). Accurately weigh, place in a stoppered conical flask, accurately add 50ml of 70% ethanol, weigh the weight, and ultrasonically treat for 40 minutes (power 560W, frequency 45kHz), let cool, weigh again, make up the lost weight with 70% ethanol, shake well, and filter through a 0.22μm filter membrane.

[0043] Asparagus cochinchinensis and Stemona bulb: Grind the product, pass through No. 3 sieve, mix well, take 0.2g, accurately weigh, place in a stoppered conical flask, accurately add 50ml of 70% ethanol, weigh the weight, ultrasonically treat for 40 minutes (power 560W, frequency 45kHz), let cool, weigh again, make up the lost weight with 70% ethanol, shake well, and filter through 0.22μm filter membrane.

[0044] 2.3 Investigation of matrix effects 2.3.1 Original dioscin reference solution: Accurately pipette 0.10 ml of original dioscin reference solution (concentration is 10.016 μg / ml) into a 10 ml volumetric flask, add 70% ethanol to dilute to the mark, and shake well to obtain the reference solution (concentration of original dioscin reference is 100.16 ng / ml).

[0045] 2.3.2 Prepare negative control solutions from the negative samples of Loquat Cough Granules, Capsules, and Soft Capsules according to the prescribed test sample solution preparation method. Accurately pipette 0.10 ml of the original dioscin reference solution (concentration: 10.016 μg / ml) into a 10 ml volumetric flask. Dilute to the mark with the negative sample solution and shake well to obtain the negative spiked solution (concentration: 100.16 ng / ml).

[0046] 2.3.3 Preparation of Stemona Radix control medicinal material solution: Take 0.2 g of Stemona erecta control medicinal material (batch number: 121588-201502, purchased from China National Institute for Food and Drug Control), Stemona foliaceae control medicinal material (batch number: 121221-201604, purchased from China National Institute for Food and Drug Control), and Asparagus cochinchinensis medicinal material (No. 13 in Table 1) according to the preparation method of the test solution in 2.2 to prepare Stemona Radix control medicinal material and Asparagus cochinchinensis medicinal material solution.

[0047] Stemona reference medicinal material spiked solution: Accurately pipette 0.10 ml of the original new dioscin reference solution (concentration is 10.016 μg / ml) into a 10 ml volumetric flask, add the Stemona solution to dilute to the scale, shake well, and the negative spiked solution (concentration of the original new dioscin reference is 100.16 ng / ml) is obtained.

[0048] 5 μl of each reference solution, Stemona control solution, negative spiked solution, and Stemona control spiked solution were accurately pipetted and injected into the liquid chromatography-mass spectrometer. The results showed negligible matrix effects, ranging from 90% to 110%. Therefore, 70% ethanol was sufficient for the preparation of reference solutions (see Tables 8-12).

[0049]

[0050]

[0051] Methodological validation 2.4.1 Specificity test Preparation of original new dioscin reference solution: Prepare according to the reference solution preparation method under 2.3.

[0052] Preparation of negative control solution: According to the prescription process of loquat cough-relieving granules, capsules and soft capsules, remove the Stemona bulbs and prepare negative samples without Stemona bulbs respectively. The negative control solutions of loquat cough-relieving granules, capsules and soft capsules are prepared according to the preparation method of the test solution.

[0053] The samples in which the original new dioscin was not detected were: Loquat cough granules: supplied by Company A, batch number: 23110903; Loquat cough capsules: supplied by Company B, batch number: S240411; Loquat cough soft capsules: supplied by Company C, batch number: 20240101; the negative sample solution was prepared according to the preparation method of the test solution.

[0054] Positive samples: Loquat cough granules: supplied by Company A, batch number: 23030901; Loquat cough capsules: supplied by Company B, batch number: S240411; Loquat cough soft capsules: supplied by Company C, batch number: 20240101; the positive sample solution was prepared according to the preparation method of the test solution.

[0055] Preparation of Stemona control medicinal material solution and Asparagus cochinchinensis medicinal material solution: Take 0.2g of Stemona erecta control medicinal material (batch number: 121588-201502, purchased from China National Institute for Food and Drug Control), and prepare Stemona control medicinal material and Asparagus cochinchinensis medicinal material solution according to the preparation method of the test solution under 3.3 for the reference medicinal material Stemona foliata control medicinal material (batch number: 121221-201604, purchased from China National Institute for Food and Drug Control) and Asparagus cochinchinensis medicinal material (No. 13 in Table 1).

[0056] Accurately aspirate 5μl of each reference solution and test solution, inject them into the HPLC-MS instrument, and determine. The results showed that no original new dioscin was detected in the negative control solution and the control preparation solution, while original new dioscin was detected in the positive sample solution. This also shows that other medicinal flavors in loquat cough granules, capsules, and soft capsules have no interference with the determination, and the specificity is strong; see Figures 1 to 9 The TIC / MRM diagrams of the reference medicinal materials of Stemona oleifera, Stemona erecta and Asparagus cochinchinensis are shown in the table. Figures 10 to 12 .

[0057] 2.4.2 Investigation of linear relationships Accurately weigh 5.008 mg of the original new dioscin reference substance and place it in a 10 ml volumetric flask. Dissolve and dilute to the mark with 70% ethanol. Shake well. Accurately pipette 2 ml and place it in a 100 ml volumetric flask. Dilute to the mark with 70% ethanol to obtain the reference substance stock solution (10.016 μg / ml). Accurately pipette 5 ml of the reference substance stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with 70% ethanol (1001.6 ng / ml). Accurately pipette 0.5 ml, 1 ml, 2.5 ml, and 5 ml of the reference substance stock solution into a 10 ml volumetric flask. Dilute to the mark with 70% ethanol to obtain curves 3 to 6. Accurately pipette 0.5 ml, 1 ml, and 2.5 ml of the solution for curve 5 into a 10 ml volumetric flask. Dilute to the mark with 70% ethanol to obtain curves 1 to 3. Accurately pipette 5 μl of the above solution and inject it into the liquid chromatography-mass spectrometer to measure the peak area. Use the injection volume of each concentration of the reference substance as the abscissa and the peak area as the ordinate to draw a standard curve and calculate the regression equation. The results are shown in Table 13. Figure 13 .

[0058]

[0059] 2.4.3 Repeatability test For granules, take the product under the item of difference in filling amount, grind it into powder, and take about 3 g; for soft capsules, take the contents under the item of difference in filling amount, mix them, and take the amount equivalent to 2 capsules (1.1 g); for Asparagus cochinchinensis, take 0.2 g of powder, weigh it accurately, place it in a stoppered conical flask, and prepare 6 test solutions according to the proposed method for preparing the test solution; for capsules, take the contents under the item of difference in filling amount, mix them, and take the amount equivalent to 2 capsules (0.5 g); for Stemona tuberosa, take 0.2 g of powder, weigh it accurately, place it in a stoppered conical flask, and accurately add 100 μl of the original dioscin solution (10.016 μg / ml), let it dry, and prepare 6 test solutions according to the proposed method for preparing the test solution.

[0060] 5 μl of each sample was accurately aspirated and injected into a liquid chromatography-mass spectrometer to determine the target component content and calculate the repeatability. The results showed that the method had good repeatability.

[0061]

[0062] 2.4.4 Precision test Precision test: Take the first sample solution under "2.4.3 Repeatability test", repeat the injection 6 times, and measure m / z The peak area of ​​the ion pair 1031.5→869.3 was calculated, and the RSD was calculated. The results showed that the method had good precision, as shown in Table 15.

[0063]

[0064] 2.4.5 Stability Investigation Take a sample solution under "2.4.3 Repeatability Test" and inject it at 0h, 4h, 8h, 16h and 24h respectively. m / z The ion pair 1031.5 → 869.3 was used for the determination. The results showed that the test solution was stable for 24 hours and met the testing requirements. The results are shown in Table 16.

[0065]

[0066] 2.4.6 Accuracy test Sample recovery reference solution: Accurately pipette 5 ml of the original new dioscin reference stock solution (see reference stock solution under 2.4.2: 10.016 μg / ml), place it into two 500 ml volumetric flasks, add 70% ethanol to dilute to the scale, shake well, and obtain sample recovery reference solution 1 (0.10016 μg / ml); accurately pipette 2.5 ml of the original new dioscin reference stock solution (see reference stock solution under 2.4.2: 10.016 μg / ml), place it into two 500 ml volumetric flasks, add 70% ethanol to dilute to the scale, shake well, and obtain sample recovery reference solution 1 (0.05008 μg / ml).

[0067] Loquat cough granules: Take the granules under the item of filling quantity difference (produced by Company A, batch number 23030901, original dioscin content 1.3268μg / g), grind it into powder, take about 3g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of original dioscin solution (0.10016μg / ml), weigh it, and ultrasonically treat it for 40 minutes (power 560W, frequency 45kHz), let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, and filter it through a 0.22μm filter membrane.

[0068] Loquat cough capsules: Take the contents of this product (produced by Company B, batch number S230817, no original dioscin was detected) under the item of filling quantity difference, grind it into powder, take about 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of original dioscin solution (0.5008μg / ml), weigh it, and ultrasonically treat it for 40 minutes (power 560W, frequency 45kHz), let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, and pass it through a 0.22μm filter membrane.

[0069] Loquat cough-relieving soft capsules: Take the granules under the item of filling quantity difference (produced by C, batch number 20240101, original dioscin content 1.0099μg / granule, average filling quantity 0.5513g / granule), grind them into powder, take about 3g, accurately weigh, place in a stoppered conical flask, accurately add 50ml of original dioscin solution (0.10016μg / ml), weigh the weight, ultrasonically treat for 40 minutes (power 560W, frequency 45kHz), let cool, weigh again, make up the lost weight with 70% ethanol, shake well, and filter through a 0.22μm filter membrane to obtain the product.

[0070] Asparagus cochinchinensis is prepared by taking 0.2 g of powder of this product (sample No. 16 in Table 1, original dioscin content 22.2851 μg / g), accurately weighing it, placing it in a stoppered conical flask, accurately adding 50 ml of original dioscin solution (0.10016 μg / ml), weighing it, and ultrasonically treating it for 40 minutes (power 560 W, frequency 45 kHz). Let it cool, and weigh it again. Make up the lost weight with 70% ethanol, shake it well, and filter it through a 0.22 μm filter membrane to obtain it.

[0071] Radix Stemonae: Take 0.2 g of powder of this product (sample No. 1 in Table 1, no original dioscin was detected), weigh it accurately, put it into a stoppered conical flask, accurately add 50 ml of original dioscin solution (0.5008 μg / ml), weigh it, and treat it with ultrasound for 40 minutes (power 560 W, frequency 45 kHz). Let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, and filter it through a 0.22 μm filter membrane.

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] 2.4.7 Detection limit Take the original new dioscin reference solution (0.2ng / ml) for analysis, see Figure 14 The signal-to-noise ratio was 3.70. Based on a signal-to-noise ratio (S / N) of 3, the detection limits of the original new dioscin were 3 ng / g (loquat cough granules), 4 ng / granule (loquat cough capsules), and 4 ng / granule (loquat cough soft capsules). 50 ng / g (Asparagus cochinchinensis and Stemona tuberosa).

[0078] 2.4.8 Durability Based on the original chromatographic conditions, an Agilent 6460 triple quadrupole mass spectrometer and an Agilent 6490 triple quadrupole mass spectrometer were used with different chromatographic columns (MicroPulite XP tC18, 1.7 μm, 2.1 × 100 mm; ThermoAcclaimTM RSLC 120 C18, 2.2 μm, 2.1 × 100 mm; NanoChromChromCore AQC18 , 1.8μm, 2.1×100mm), samples (granules: produced by Company A, batch number 23030901; capsules: produced by Company B, batch number S230817, no original dioscin was detected, about 0.5g was taken, accurately weighed, placed in a stoppered conical flask, and 50ml of original dioscin solution (0.5008μg / ml) was accurately added for determination; soft capsules: produced by Company C, batch number 20240101), Stemona japonica (sample No. 1 in Table 1), and Asparagus cochinchinensis (sample No. 16 in Table 1) were determined to examine the durability of the instrument and chromatographic column. The results are shown in Tables 22-23.

[0079]

[0080]

[0081] An Agilent 6460 triple quadrupole mass spectrometer and an Agilent 6490 triple quadrupole mass spectrometer and different chromatographic columns (MicroPulite XP tC18, 1.7μm, 2.1×100mm; Thermo AcclaimTM RSLC 120C18, 2.2μm, 2.1×100mm; NanoChromChromCore AQC18, 1.8μm, 2.1×100mm) were investigated. The results showed that both brands and models of instruments and three different brands of chromatographic columns were capable of determining protodioscin, indicating that the instrument and chromatographic columns used in this method are robust.

[0082] 3. Sample Measurement 3.1 Limit setting Prepare test solutions from the medicinal materials of Stemona radix and Asparagus cochinchinensis according to the test solution preparation method under 2.2. Using the prescribed chromatographic and mass spectrometry conditions, precisely pipette 5 μl of each sample (some of the original dioscin in the Asparagus cochinchinensis medicinal material was diluted 10-fold with the test solution before injection) and inject into a high-performance liquid chromatography-mass spectrometer to determine the content of original dioscin in Stemona radix and Asparagus cochinchinensis. Results showed that original dioscin was not detected in any of the 15 batches of Stemona radix. The content of original dioscin in 26 batches of Asparagus cochinchinensis ranged from 8.08 μg / g to 1945.27 μg / g. A normal distribution curve was drawn for the original dioscin content in Asparagus cochinchinensis, and the average value within the 95% confidence interval was 227.55 μg / g. For details, see Table 24 below.

[0083] The limit of adulteration of Asparagus cochinchinensis in Radix Stemonae: The General Rules for the Verification of Medicinal Materials and Decoction Pieces of Part IV 0212 of the 2020 edition of the Chinese Pharmacopoeia stipulates that "drug scraps and impurities shall not usually exceed 3%." There are cases of unintentional adulteration and the mixing of a small amount of Asparagus cochinchinensis into Radix Stemonae. Therefore, in order to prevent misjudgment and target the case of subjective adulteration, the limit is calculated for samples after 5% Asparagus cochinchinensis is added to Radix Stemonae. According to the average content of original dioscin in Asparagus cochinchinensis medicinal materials of 227.55μg / g, the limit of Radix Stemonae medicinal materials is calculated based on the addition of 5% Asparagus cochinchinensis. The result is that this product contains original dioscin (C 51 H 84 O 22 ) shall not exceed 11.38μg / g. The test solution is prepared according to the proposed preparation method. When the concentration of original dioscin in the test solution is 46ng / ml, the upper limit is reached.

[0084] Limits for loquat cough suppressant preparations: Based on the prescription quantity and the [Preparation Method] section, Loquat Cough Relief Granules (50g of Stemona Root, made into 1000g), Loquat Cough Relief Capsules (75g of Stemona Root, made into 1000 capsules), and Loquat Cough Relief Soft Capsules (75g of Stemona Root, made into 1000 capsules), the limits were calculated based on an average raw dioscin content of 227.55 μg / g in Asparagus cochinchinensis, assuming a 5% asparagus incorporation and a 100% transfer rate. The results are: 0.57 μg / g for Loquat Cough Relief Granules; 0.85 μg / capsule for Loquat Cough Relief Capsules; and 0.85 μg / capsule for Loquat Cough Relief Soft Capsules. Prepare the test solution according to the proposed preparation method. The upper limit of the raw dioscin limit was reached when the test solution reached 34.2 ng / ml. Taking into account the large differences in process technology among manufacturers, the limit is appropriately relaxed to 40ng / ml, and any amount exceeding the limit is considered as detection.

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[0086]

[0087] 4.2 Result determination In summary, the proposed judgment principles are: Judgment principles (1) If the chromatographic peak corresponding to the reference solution chromatogram does not appear simultaneously in the extracted ion current chromatogram of the test sample, it is considered not detected; (2) If the chromatographic peak corresponding to the reference solution chromatogram appears simultaneously in the extracted ion current chromatogram of the test sample, and the chromatographic peak area value of m / z 1031.5→869.3 in the chromatogram of the test sample is not greater than the corresponding peak area value in the reference solution, it is considered not detected; (3) If the chromatographic peak corresponding to the reference solution chromatogram appears simultaneously in the extracted ion current chromatogram of the test sample, and the chromatographic peak area value of m / z 1031.5→869.3 in the chromatogram of the test sample is greater than the corresponding peak area value in the reference solution, it is considered detected.

[0088] Results Determination: In the extracted ion current chromatogram of the test sample, no chromatographic peak corresponding to the chromatogram of the reference solution should be detected.

[0089] 5. Sample measurement and results According to the proposed method, 175 batches of loquat cough granules and capsules collected in the national drug random inspection in 2024 were inspected. The results showed that the content of original new dioscin in 2 batches of loquat cough granules samples exceeded the proposed limit, with a detection rate of 1.1%. The original new dioscin component was also detected in another 5 batches of loquat cough granules, but it did not exceed the limit. Typical chromatograms are shown in Figure 2. Figure 15 .

Claims

1. A method for detecting adulteration of Asparagus cochinchinensis in a Radix Stemonae Radix and a loquat cough suppressant preparation containing the Radix Stemonae Radix raw material, characterized by: The loquat cough-relieving preparation includes loquat cough-relieving granules, loquat cough-relieving capsules and loquat cough-relieving soft capsules. The determination method comprises the following steps: (1) Chromatographic, mass spectrometric conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, and 0.1% formic acid 10 mmol / L ammonium formate solution was used as the mobile phase B. Gradient elution was performed as specified in the following table; the flow rate was 0.25 ml / min; the column temperature was 35 °C; the mass spectrometer was used in the electrospray positive ionization mode (ESI + ), multiple reaction monitoring (MRM); select mass-to-charge ratio m / z 1031.5→869.3 and m / z 1031.5→577.3 is used as the detection ion pair for original dioscin; 5 μl of the reference solution is injected, and the signal-to-noise ratio of the MRM chromatographic peak measured by the above detection ion pair should be greater than 10:1; (2) Preparation of reference solution Reference solution of Radix Stemonae Radix: Take an appropriate amount of original new dioscin protoneodioscin reference substance, accurately weigh it, place it in a 50ml volumetric flask, add 70% ethanol to dissolve it and dilute it to the scale, shake it well, and then dilute it with 70% ethanol to a solution containing 46ng per 1ml. Reference solution of loquat cough suppressant preparation: Take an appropriate amount of original new dioscin protoneodioscin reference substance, accurately weigh it, place it in a 50ml volumetric flask, add 70% ethanol to dissolve it and dilute it to the scale, shake it well, and then dilute it with 70% ethanol to a solution containing 35ng per 1ml. (3) Preparation of test solution Stemona tuberosa test solution: grind the product, pass through a No. 3 sieve, mix well, take 0.2 g, accurately weigh, place in a stoppered conical flask, accurately add 50 ml of 70% ethanol, weigh the weight, ultrasonicate for 40 minutes, let cool, weigh again, make up the lost weight with 70% ethanol, shake well, and filter through a 0.22 μm filter membrane. Loquat cough suppressant test solution: For granules, take the product listed under "Difference in Filling Amount", grind it into powder, take 3 g, and accurately weigh. For capsules and soft capsules, take the product listed under "Difference in Filling Amount", mix thoroughly, take an amount equivalent to 2 capsules, and accurately weigh. Place in a stoppered conical flask, accurately add 50 ml of 70% ethanol, weigh the solution, ultrasonicate for 40-45 minutes, cool, weigh again, make up the loss with 70% ethanol, shake well, filter, and obtain the filtrate. (4) Determination: Accurately pipette 1-5 μl of the reference solution and the test solution, inject them into the high performance liquid chromatography-mass spectrometer, measure, and record the chromatogram; (5) Judgment principle: ① If the chromatographic peak corresponding to the chromatographic peak of the reference solution does not appear in the extracted ion current chromatogram of the test sample, it is considered as not detected; ② If the chromatographic peak corresponding to the chromatographic peak of the reference solution appears in the extracted ion current chromatogram of the test sample, and the chromatographic peak of the reference solution appears in the extracted ion current chromatogram of the test sample, m / z If the chromatographic peak area value of 1031.5→869.3 is not greater than the corresponding peak area value in the reference solution, it is considered as not detected; ③ In the extracted ion current chromatogram of the test sample, the chromatographic peak corresponding to the reference solution chromatogram appears at the same time, and the chromatographic peak of the test sample is m / z If the chromatographic peak area value of 1031.5→869.3 is greater than the corresponding peak area value in the reference solution, it is considered detected; (6) Result determination: In the extracted ion current chromatogram of the test sample, no chromatographic peak corresponding to the chromatogram of the reference solution should be detected.

2. The method for detecting adulteration of Asparagus cochinchinensis in the Radix Stemonae and loquat cough-relieving preparations containing the Radix Stemonae according to claim 1, characterized in that: In the preparation of the test solution in step (3), the ultrasonic treatment has an ultrasonic power of 560 W and a frequency of 45 kHz.

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